Simian-human HAV having a chimeric 2C protein

ABSTRACT

The present invention discloses simian-human hepatitis A virus chimeric genomes which encode a hepatitis A virus having a chimeric 2C protein. The invention further discloses the use of these viruses or the nucleic acid sequence encoding them as vaccines.

This application claims the benefit of International Application No.PCT/US97/06506, filed on Apr. 18, 1997, and U.S. Provisional ApplicationNo. 60/015,642 filed Apr. 19, 1996.

FIELD OF THE INVENTION

The present invention relates to molecular approaches to the developmentof a live hepatitis A vaccine. In particular, the invention relates tonucleic acid sequences which encode hepatitis A viruses having achimeric 2C protein. More specifically, the nucleic acid sequences ofthe invention comprise a genome of a human hepatitis A virus strainwhich contains a chimeric 2C gene consisting of sequences from both ahuman strain and the simian AGM-27 strain. The invention further relatesto the use of these viruses, or the nucleic acid sequences encodingthem, as vaccines.

BACKGROUND OF THE INVENTION

Hepatitis A virus (HAV) is a picornavirus with a ˜7.5 kb positive strandRNA genome and is the sole member of the Hepatovirus genus (Francki, R.I. B., et al. (1991) Classification and Nomenclature of Viruses. (Arch.Virol./Suppl. 2). Springer, Vienna). The clinical manifestations of HAVinfection in humans can vary greatly, ranging from asymptomaticinfection, commonly seen in young children, to fulminant hepatitis,which in some cases can result in death (Ross, B. C., et al. (1991) Adv.Virus Res., 39:209-253).

In attempting to prevent hepatitis A, three general strategies arepossible: 1) increasing hygiene standards; 2) passive immunization ofthose known to be exposed to HAV with normal human immune globulin; and3) the development of HAV vaccines. However, because sanitation levelsin underdeveloped countries remain low and passive immunization offerslittle hope for control of endemic hepatitis A since most cases ofhepatitis A occur in individuals who do not have a specific exposurehistory, considerable research efforts have been devoted to thedevelopment of either live or killed vaccines.

With respect to killed or inactivated vaccines, numerous laboratorieshave reported the development of inactivated HAV vaccines (see, forexample, Binn, L. N. et al. (1986) J. Inf. Dis., 153:749; Provost, P. J.et al. (1986) J. Med. Virol., 19:23; Flehmig, B. et al. (1989) Lanceti:1039 and Andre, F. E. et al. (1990) Progress in Med. Virol., 37:72)and SmithKline Beecham and Merck have recently licensed and soldinactivated HAV vaccines containing different strains of HAV. However,the high cost of inactivated HAV vaccines makes their use in other thanhigh-risk individuals unlikely. In addition, questions concerning theduration of immunity induced by inactivated HAV vaccines suggests thatmultiple doses may need to be administered to confer continuedprotection. Thus, for these reasons, the widespread use of liveattenuated HAV vaccines in underdeveloped countries where hepatitis A isendemic may be more feasible and more efficacious than use ofinactivated vaccines.

In attempting to develop a live attenuated vaccine, numerousinvestigators have selected attenuated hepatitis A viruses by passage ofwild-type HAV strains in cell culture (see, for example, Provost et al.(1986) J. Med. Virol., 20:165-176; Karron, R. A. et al. (1988) J.Infect. Dis., 157:338-345). However, attenuation of HAV strains duringadaptation to growth in cell culture has been observed to result inoverattenuation such that the attenuated viruses, when administered aslive vaccines,are no longer effective inducers of anti-HAV antibodies invivo (Provost, P. J. et al. (1986) J. Med. Virol., 20: 165-175).

A potential alternative approach to the production of a candidate liveattenuated vaccine strain which grows sufficiently well in a cell lineto make vaccine production economically feasible and which is alsoinfectious, immunogenic and avirulent in humans, is the use ofrecombinant DNA methodology to construct chimeric HAV genomes.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid sequences which comprise agenome of a human hepatitis A strain which contains a chimeric 2C geneconsisting of sequences from both the human strain and the simian AGM-27strain. The nucleic acid sequences of the invention are designated “2Cchimeric genomes”.

It is therefore an object of the invention to provide nucleic acidsequences which encode hepatitis A viruses having a chimeric 2C protein.For the purposes of this application, nucleic acid sequence refers toRNA, DNA, CDNA or any variant thereof capable of directing host organismsynthesis of hepatitis A viruses having a chimeric 2C protein.

The invention also relates to hepatitis A viruses encoded by the 2Cchimeric genomes. These viruses are designated “2C chimeric hepatitis Aviruses.”

The invention further provides vaccines for use in immunizing a mammalagainst hepatitis A. In one embodiment, the vaccine comprises a 2Cchimeric hepatitis A virus. In a second embodiment, the vaccinecomprises a 2C chimeric genome which encodes a hepatitis A virus havinga chimeric 2C protein.

The invention therefore also relates to methods for preventing hepatitisA in a mammal. In one embodiment, the method comprises administering toa mammal an amount of a 2C chimeric genome of the invention effective toinduce protective immunity against hepatitis A. In another embodiment,the method of prevention comprises administering to a mammal a 2Cchimeric hepatitis A virus in an amount effective to induce protectiveimmunity against hepatitis A.

The invention also provides pharmaceutical compositions comprising the2C chimeric genome of the invention and/or their encoded hepatitis Aviruses.

The invention further provides kits comprising the 2C chimeric nucleicacid sequences of the invention.

The invention further relates to antibodies to 2C chimeric hepatitis Aviruses and to pharmaceutical compositions comprising these antibodies.

DESCRIPTION OF FIGURES

FIG. 1 shows the genomic structure of full-length cDNA clones ofchimeras consisting of HAV/7 (white), a cell culture-adapted variant ofwild-type HM-175 HAV derived by passage of HM-175 35 times in primaryAfrican Green Monkey kidney cells (see Table 1 of Cohen et al (1987)Proc. Nat'l Acad. Sci USA, 84:2497-2501 for HAV/7 sequence), wild-typeHM-175 (black) (see FIG. 1 of Cohen et al (1987) J. Virol., 61:50-59 forwild-type HM-175 sequence) and AGM-27 (grey) (see SEQ. ID. NO:1 of U.S.Pat. No. 5,476,658 for AGM-27 sequence) sequences. The chimeras wereconstructed using DNA fragments from molecular clones of HAV/7 andwild-type HM-175 and fragments of PCR amplified cDNA from the AGM-27virus. The 2A/2B junctions in the diagrams presented in this applicationcorrespond to the cleavage site designation of Cohen et al. (1987) (J.Virol., 61:50-59) at nucleotides 3674/3675. However, recent results haveshown that the 2A/2B junction is in fact nucleotides 3242/3243. Thus,when reference is made to the “2B gene” or “2B sequence” in thisapplication it refers to the 2B gene as defined by Cohen et al. (J.Virol. 61:50-59) which is in reality a truncated 2B which consists ofapproximately 42% of the residues at the 3′ end of the 2B gene.

FIG. 2 presents a schematic diagram showing the amino acid sequencedifferences in the proteins between wild-type HM-175 and AGM-27. Thebars mark positions at which the amino acid sequence differs betweenAGM-27 and wild-type HM-175. There are 30 amino acid differences betweenthe 2C sequences of wild-type HM-175 and AGM-27. Cell culture adaptationof HM-175 to produce HAV/7 resulted in three amino acid changes in 2C atresidues 31, 76 and 190. When the comparison is between HAV/7 andAGM-27, there are 31 amino acid differences. Amino acid residues 31 and190, which are the same in wild-type HM-175 and AGM-27, differ betweenHAV/7 and AGM-27. Amino acid residue 76, which differs between wild-typeHM-175 and AGM-27, is the same in HAV/7 and AGM-27. The asterisks markthe sites of the three mutations that are critical for growth of HAV/7in cell culture (nts 3889 in 2B and 4087 and 4222 in 2C) (Emerson, S.U., et al. (1992) J. Virol., 66:650-654). The locations of the twoconserved putative NTP-binding motifs are shown with arrows. The aminoacid sequence differences in 2C between HM-175 (either wild-type orHAV/7) and AGM-27 are clustered at the amino-terminal andcarboxy-terminal ends of the protein. The mutation at position 3889 inthe 2B gene, which greatly enhances the growth of human HAV in cellculture, is absent in AGM-27.

FIG. 3 shows the results of a radioimmunofocus assay comparing the sizesof the foci formed by HAV/7, AGM-27 and the GR2 chimera which containsthe AGM-27 2C sequence in the HAV/7 background.

FIGS. 4A-4F shows the results of a radioimmunofocus assay comparingfocus size of chimeras that differ in the truncated 2B gene sequenceand/or the 2C gene sequence. (A) HAV/7, (B) GR2, (C) GR3, (D) GR4, (E)GR15 and (F) GR9.

FIG. 5 shows a hybridization assay comparing the growth rates of HAVchimeras which differ only in the sequence of the 2C gene. The chimerascontained either the cell culture-adapted HM-175 (HAV/7), wild-typeHM-175 (36Y) or AGM-27 2C sequence in the HAV/7 background (GR2). ViralRNA was quantified by slot blot hybridization followed byautoradiography and densitometry analysis. In the bar diagramidentifying the virus genotype, AGM-27 sequences are shaded in grey andthe four boxes shown in each diagram indicate from left to right(5′→3′): HAV/7 genomic sequence 5′ of the truncated 2B gene, truncated2B gene, 2C gene and HAV/7 genomic sequence 3′ of the 2C gene. Thedemarcations of the truncated 2B gene and of the 2C gene as shown in thebar diagrams are not drawn to scale.

FIG. 6 shows results of a hybridization assay of growth curves of HAV/7,the simian-human chimera GR2, and the intragenic 2C chimeras, GR3 andGR4. Viral RNA was quantified by slot blot hybridization followed byautoradiography and densitometry analysis. In the bar diagramidentifying the virus genotype, AGM-27 sequences are shaded in grey andthe four boxes shown in each diagram indicate from left to right(51′→3′): HAV/7 genomic sequence 5′ of the truncated 2B gene, truncated2B gene, 2C gene and HAV/7 genomic sequence 3′ of the 2C gene. Thedemarcations of the truncated 2B gene and of the 2C gene as shown in thebar diagrams are not drawn to scale.

FIG. 7 shows the results of a hybridization assay of growth curves ofHAV/7 and chimeras containing the truncated AGM-27 2B gene (GR15), the2C gene (GR2) or the truncated 2B gene and the 2C gene (GR9) in theHAV/7 background. Viral RNA was quantified by slot blot hybridizationfollowed by autoradiography and densitometry analysis. In the bardiagram identifying the virus genotype, AGM-27 sequences are shaded ingrey and the four boxes shown in each diagram indicate from left toright (5′→3′): HAV/7 genomic sequence 5′ of the truncated 2B gene,truncated 2B gene, 2C gene and HAV/7 genomic sequence 3′ of the 2C gene.The demarcations of the truncated 2B gene and of the 2C gene as shown inthe bar diagrams are not drawn to scale.

FIGS. 8A and 8B show biochemical (ICD levels), serological (anti-HAV),histopathological and PCR analyses of two tamarins 782 (FIG. 8A) and 783(FIG. 8B) inoculated with the GR2 chimera which contains the AGM-27 2Cgene in the HAV/7 background. The ICD serum enzyme levels were measuredin international units per ml (IU/ml). A + in the row marked “anti-HAV”indicates samples that were positive for anti-HAV antibodies asdetermined by commercial assay. The histopathology scores correspond tomild hepatitis (1+), mild to moderate hepatitis (2+), moderately severehepatitis (3+) and severe hepatitis (4+). The “ND” designation signifiesthat liver histology analysis was not performed for these samples. Forthe PCR analyses, an open circle indicates that the stool sampleanalyzed was completely negative for HAV after two steps of nested PCR;a half-closed circle indicates that the stool sample was positive forHAV after two steps of nested PCR and a closed circle indicates that thestool sample was positive for HAV after one step of PCR.

FIGS. 9A and 9B show biochemical (ICD levels) serological (anti-HAV),histopathological and PCR analyses of two tamarins, 808 (FIG. 9A) and790 (FIG. 9B), inoculated with the GR3 chimera which contains sequencefrom nt 3996-4357 of the AGM-27 2C gene in the HAV/7 background. The ICDserum enzyme levels were measured in international units per ml (IU/ml).A + in the row marked “anti-HAV” indicates samples that were positivefor anti-HAV antibodies as determined by commercial assay. The “ND”designation signifies that liver histology analysis was not performedfor these samples. For the PCR analyses, an open circle indicates thatthe stool sample analyzed was completely negative for HAV after twosteps of nested PCR; a half-closed circle indicates that the stoolsample was positive for HAV after two steps of nested PCR and a closedcircle indicates that the stool sample was positive for HAV after onestep of PCR.

FIGS. 10A and 10B show biochemical (ICD levels), serological (anti-HAV),histopathological and PCR analyses of two tamarins, 799 (FIG. 10A) and818 (FIG. 10B), inoculated with the GR4 chimera which contains sequencefrom nt 4354-4981 of the AGM-27 2C gene in the HAV/7 background. The ICDserum enzyme levels were measured in international units per ml (IU/ml).A + in the row marked “anti-HAV” indicates samples that were positivefor anti-HAV antibodies as determined by commercial assay. Thehistopathology scores correspond to mild hepatitis (1+), mild tomoderate hepatitis (2+), moderately severe hepatitis (3+) and severehepatitis (4+). The “ND” designation signifies that liver histologyanalysis was not performed for these samples. For the PCR analyses, anopen circle indicates that the stool sample analyzed was completelynegative for HAV after two steps of nested PCR; a half-closed circleindicates that the stool sample was positive for HAV after two steps ofnested PCR and a closed circle indicates that the stool sample waspositive for HAV after one step of PCR.

FIGS. 11A-11D show biochemical, serological (anti-HAV) andhistopathological analyses of two chimpanzees, 1558 (FIGS. 11A and 11C)and 1564 (FIGS. 11B and 11D), inoculated with the GR4 chimera. In FIGS.11A and 11B, the biochemical responses are shown as ICD levels where theICD serum enzyme levels were measured in international units per ml(IU/ml). In FIGS. 11C and 11D, the biochemical responses are shown asALT levels where the ALT levels were measured in international units/ml.A + in the row marked “anti-HAV” indicates samples that were positivefor anti-HAV antibodies as determined by commercial assay. Thehistopathology scores correspond to mild hepatitis (1+), mild tomoderate hepatitis (2+), moderately severe hepatitis (3+) and severehepatitis (4+). The “ND” designation signifies that liver histologyanalysis was not performed for these samples.

FIGS. 12A and 12B show biochemical (ALT levels), serological (anti-HAV)and histopathological analyses of two chimpanzees 1545 (FIG. 12A) and1547 (FIG. 12B) inoculated with the GR2 chimera. A + in the row marked“anti-HAV” indicates samples that were positive for anti-HAV antibodiesas determined by commercial assay. The histopathology scores correspondto mild hepatitis (1+), mild to moderate hepatitis (2+), moderatelysevere hepatitis (3+) and severe hepatitis (4+). The “ND” designationsignifies that liver histology analysis was not performed for thesesamples.

FIGS. 13A-13G show the complete nucleotide (SEQ ID NO: 1) and predictedamino acid sequences for wild-type HAV HM-175.

FIG. 14 shows the results of a comparison between the genome sequencesof wild-type (WT) HAV HM-175 (shown in FIGS. 13A-13D) and attenuated(attenuated) cell-culture adapted HAV/7 where the difference between thetwo genomes are indicated in the columns marked WT and attenuated.Nucleotide positions correspond to numbering for wild-type HM-175 shownin FIGS. 13A-13D.

FIGS. 15A-15E show the nucleotide sequence of greater than 99% of theentire genome of AGM-27 (SEQ ID No: 3). The sequence determined forAGM-27 starts from nucleotide 59 according to the nomenclature forwild-type HM-175.

DESCRIPTION OF INVENTION

The present invention relates to nucleic acid sequences which encode ahepatitis A virus having a chimeric 2C protein. More specifically, theinvention relates to nucleic acid sequences which comprise a genome ofhuman hepatitis A strain having a chimeric 2C gene which consists ofsequences from both the human strain and the simian AGM-27 strain. In apreferred embodiment, the human hepatitis A strain is an attenuatedhepatitis A strain such as MRC5 or HAV/7. In a more preferredembodiment, the attenuated human hepatitis A strain is HAV/7 (Cohen etal. (1987) Proc. Natl. Acad. Sci. USA, 84:2497-2501). Thus, in apreferred embodiment, the 2C chimeric genomes of the invention comprisea genome of the HAV/7 strain having a chimeric 2C gene which consists ofsequences from both the HAV/7 strain and the simian AGM-27 strain.

Chimeras of the invention can be generated from the pGRI, pGR2 andpHAV/7 full-length clones presented herein in the Examples sectionthrough the use of PCR and cloning techniques.

Chimeras of the invention include chimeras 1-18, which are generated byPCR amplification of HAV/7 sequence using primers with engineeredrestriction sites followed by subcloning into the pGR2 background.

# AGM-27- specific 2C AGM-27 2C Amino Amino Acid HAV/7 Region AcidSequence Residues Chimera # Amplified in Chimera in Chimera 1 PpuM1 toDraI residues 48-328 23 2 PpuM1 to Bc1I residues 63-328 20 3 PpuM1 toresidues 84-328 18 XmnI 4 PpuM1 to Sal I residues 98-328 16 5 DraI toAflII residues 1-49 and 121-328 20 6 BclI to AflII residues 1-64 and121-328 24 7 XmnI to AflII residues 1-86 and 121-328 26 8 SalI to AflIIresidues 1-99 and 121-328 27 9 DraI to EcoR1 residues 1-49 7 10 BclI toEcoR1 residues 1-64 11 11 XmnI to EcoR1 residues 1-86 13 12 SalI toEcoR1 residues 1-99 14 13 HinfI to EcoR1 residues 1-283 22 14 AflIII toEcoR1 residues 1-294 24 15 BalI to EcoR1 residues 1-304 25 16 AflII toHinfI residues 1-120 and 283-328 25 17 AflII to AflIII residues 1-120and 294-328 23 18 AflII to BalI residues 1-120 and 303-328 22

Restriction sites used in AGM-27 for generating these chimeras are:

DraI nucleotides 4136-4141 BclI nucleotides 4181-4186 XmnI nucleotides4245-4254 SalI nucleotides 4287-4292 HinfI nucleotides 4841-4845 AflIIInucleotides 4873-4878 BalI nucleotides 4901-4906 PflM1 nucleotides4205-4215

All these sites, with the exception of PflM1, are present in the AGM-27sequence but not in the HAV/7 sequence. These restriction sites areengineered at the analogous positions in the pHAV/7 2C gene byprimer-directed mutagenesis using PCR. Fragments of pHAV/7 2C sequenceare amplified by PCR using the primers with engineered restriction sitesand the PCR products are then digested with the appropriate restrictionenzymes and cloned into the pGR2 background. Where the restrictionenzymes to be utilized in constructing the chimeras of the inventionrecognize multiple sites in the HAV/7 genome, one can subclone the 2Cgene into a suitable vector prior to generation of the chimeras.Suitable vectors include, but are not limited to, plasmids, PUC vectors(Gibco-BRL), pCRII (Invitrogen), pGEM vectors (Promega) and pBS vectors(Stratagene).

Additional chimeras of the invention include chimeras 19-21, which aregenerated by substituting portions of the AGM-27 2C gene for thecorresponding portion of the HAV/7 2C gene in, for example, the pGR1construct.

# AGM-27- AGM-27 2C Amino specific 2C Amino pGR2 Region Acid SequenceAcid Residues Chimera # Amplified in Chimera in Chimera 19 PpuM1 toPflM1 residues 1-70 12 20 PflM1 to AflII residues 74-120  5 21 PflM1 toEcoR1 residues 74-328 18

Chimeras 19-are constructed by engineering a PflM1 site into the AGM-272C gene. The appropriate segment of the pGR2 2C gene is then amplifiedby PCR using a primer with an engineered PflM1 restriction site and theresulting PCR product is digested with the appropriate enzymes andsubcloned into the pGR1 background.

Since the HAV 2C gene encodes 335 amino acids, it is understood by thoseof ordinary skill in the art that the amino acid residues in thechimeras of the invention that are not from AGM-27 are from HAV/7. Thusfor example, in chimera #19, 2C amino acids 1-70 are from AGM-27 andamino acids 71-335 are from HAV/7. In one embodiment the chimeras of thepresent invention encode at least one and no greater than thirty one ofthe AGM-27 2C amino acid residues which are different from the aminoacids present in the corresponding amino acid sequence of HAV/7; in amore preferred embodiment, the chimeras encode between about 5 to about25 of the AGM-27 2C amino acid residues which are different from theamino acids present in the corresponding amino acid sequence of HAV/7;and in a most preferred embodiment, the chimeras encode between about 10to about 20 of the AGM-27 2C amino acid residues which are differentfrom the amino acids present in the corresponding amino acid sequence ofHAV/7.

In addition, the column marked “#AGM-27-specific 2C Amino Acid Residuesin Chimera” indicates the number of AGM-27 2C amino acid residuespresent in the chimera which are different from the amino acid residuespresent in the corresponding amino acid sequence of HAV/7.

In an alternative embodiment, 2C chimeric genomes of the invention canbe generated using fusion recombinant PCR techniques. For example, afragment containing HAV/7 sequence corresponding to sequences upstreamof the PpuM1 site to any nucleotide in the 2C gene can be generated byPCR amplification of HAV/7 plasmid DNA using appropriate primers. Thereverse primer in this reaction would have 5′-add-on sequencescorresponding to AGM-27 sequences just 3′ to the gene fusion junction.Similarly, AGM-27 sequence corresponding to sequences from the desiredgene fusion junction to sequences downstream of the EcoR1 restrictionsite near the 3′ end of the 2C gene can be generated using plasmid pGR2as a template. The forward primer in this reaction would have 5′-add-onsequences corresponding to HAV/7 sequences just 5′ to the gene fusionjunction. This primer can be the complement of the reverse primer usedfor amplification of the HAV/7 sequence. The products of these two PCRreactions would have overlapping sequence which would include the genefusion junction. The overlapping sequence can be extended by a DNApolymerase to generate a product that is the sum of the two overlappingfragments (i.e. the 2C gene from 5′ of the PpuM1 site to sequences just3′ of the EcoR1 site). This gene fusion PCR product can be amplified bystandard PCR; the forward primer in this reaction may include the PpuM1site and the reverse primer may include the EcoR1 site. The PCRamplified fragment containing the intact hybrid 2C gene would then bedigested with PpuM1 and EcoR1 and subcloned into a modified HAV/7 clone(e.g. 32Y or pGR1 or pGR2). Using such gene fusion recombinant PCRtechniques, chimeras which contain, for example, AGM-27 sequence fromamino acid 90-328 (which includes 17 of the 31 amino acid differences in2C between HAV/7 and AGM-27) or AGM-27 sequence from amino acid 267-328(which includes 10 of the 31 amino acid differences in 2C between HAV/7and AGM-27) can be generated.

Alternatively, chimeras containing different amounts of AGM-27 sequenceat the amino-terminal end of the 2C protein can also be generated usingsimilar techniques; in this case, the fragment containing the PpuM1 sitewould be amplified by PCR using pGR2 as a template and the fragmentcontaining the EcoR1 site would be amplified using HAV/7 as a template.Of course, those of ordinary skill in the art would readily understandthat the chimeras of the present invention could also be produced byother techniques common to molecular biology such as site-directedmutagenesis.

The present invention further relates to the production of 2C chimericviruses from the nucleic acid sequences described herein.

In one embodiment, the 2C chimeric genomes of the invention can beinserted into an expression vector that functions in eukaryotic cells.Examples, of such vectors include, but are not limited to, plasmidexpression vectors and vaccinia virus vectors.

The 2C chimeric genome contained in the recombinant expression vectorcan also be transcribed in vitro by methods known to those of ordinaryskill in the art in order to produce RNA transcripts which encode the 2Cchimeric hepatitis A viruses of the invention. The 2C chimeric hepatitisA viruses of the invention may then be produced by transfecting cells bymethods known to those of ordinary skill in the art with either the invitro transcription mixture containing the RNA transcripts or with therecombinant expression vectors containing the 2C chimeric genome. Suchmethods include, but are not limited to, electroporation, andlipofection and transfection with DEAE-dextran. Cells suitable for invitro transfection with the RNA transcripts and recombinant expressionvectors of the present invention include eukaryotic cell lines, cellsput into primary culture from a host, or cells resulting from passage ofthe primary culture. Examples of preferred cells are MRC-5, AGMK, FRhK-4and BSC-1 cells.

The 2C chimeric virus so generated can be tested for virulence phenotypeby administering the 2C chimeric virus to tamarins and examining thelivers of the tamarins for evidence of pathology and/or the serum forbiochemical evidence of hepatitis as measured by levels of liver enzymessuch as isocitrate dehydrogenase and alanine aminotransferase and theviruses can be tested for their growth in cell culture by techniquessuch as RIFA or slot blot hybridization as described in Examplessection. The 2C chimeric viruses produced from the chimeric sequences ofthe invention may be purified or partially purified from the transfectedcells by methods known to those of ordinary skill in the art such asthose described in Andre et al. (Prog. Med. Virol., (1990) 37:72-95) andProvost et al. (J. Med. Virol., (1986) 19:23-20, both of which arehereby incorporated by reference. In a preferred embodiment, the 2Cchimeric viruses are partially purified prior to their use as immunogensin the pharmaceutical compositions and vaccines of the presentinvention.

The present invention therefore relates to the use of 2C chimericviruses as immunogens in live vaccines to prevent hepatitis A in amammal. When used as a live vaccine, the 2C chimeric virus can beadministered alone or in a suitable diluent such as saline or water. Thevaccine of the invention may be administered to the mammal by a varietyof routes including, but not limited to, orally, subcutaneously,intramuscularly or intravenously. A preferred route of administration isorally. Suitable amounts of chimeric hepatitis A virus may range fromabout approximately 10³ to about 10⁸ tissue culture infectious doses(TCID), more preferably, from about 10⁴ to about 10⁷ TCID. Those ofordinary skill in the art would readily understand that suitableconcentrations of 2C chimeric virus to include in the vaccines of theinvention will vary depending on the route of administration chosen. Theimmunogens of the invention may be administered once or at periodicintervals until a protective titer of anti-HAV antibody is produced.

In a preferred embodiment, the vaccine of the invention is administeredto mammals selected from the group consisting of humans, apes andmonkeys.

In an alternative embodiment, the immunogen of the present invention maybe a nucleic acid sequence which encodes a 2C chimeric HAV. Where thesequence is a cDNA sequence, the cDNAs and their RNA transcripts may beused to transfect a mammal by direct injection into the liver tissue ofthe mammal (Emerson, S. U. et al. (1992) J. Virol., 66:6649-6654,incorporated herein by reference).

Alternatively, direct gene transfer may be accomplished viaadministration of a eukaryotic expression vector containing a nucleicacid sequence of the invention.

Suitable routes of administration for a nucleic acid immunogen include,but are not limited to, intramuscular, subcutaneous or intradermaladministration. Eukaryotic expression vectors suitable for producinghigh efficiency gene transfer in vivo are known to those of ordinaryskill in the art and include, but are not limited to, plasmid-basedexpression vectors and retroviral and adenoviral vectors.

Doses of nucleic acid sequence effective to elicit a protective antibodyresponse against hepatitis A range from about 250 μg to about 5 mg, morepreferably from about 1 mg to 2 mg.

The 2C chimeric viruses and the nucleic acid sequences encoding theseviruses may be supplied in the form of a kit, alone, or in the form of apharmaceutical composition.

The administration to mammals of either the 2C chimeric genomes or the2C chimeric viruses of the invention may be for either a prophylactic ortherapeutic purpose. When provided prophylactically, the viruses ornucleic acid sequences are provided in advance of any exposure to HAV orin advance of any symptom due to HAV infection. The prophylacticadministration therefore serves to prevent or attenuate any subsequentinfection of the mammal with HAV. When provided therapeutically, theviruses or nucleic acid sequences are provided at, or shortly after, theonset of infection or disease caused by HAV. The therapeuticadministration of the viruses or nucleic acid sequences of the inventionthus attenuates the infection or disease.

In addition to use as a vaccine, the 2C chimeric genomes and 2C chimericviruses of the invention can be used to prepare antibodies to HAV. Theseantibodies can be used directly as antiviral agents or they may be usedin immunoassays such as ELISA, Western blotting and immunohistochemistryto detect HAV or HAV proteins.

The antibodies of the present invention may be contained in antiserumobtained from a mammal immunized with the 2C chimeric genomes or the 2Cchimeric viruses of the invention. Alternatively the antibodies may bepolyclonal antibodies purified or partially purified from the antiserumor monoclonal antibodies. The antibodies of the invention may beutilized for pre- or post-exposure passive immunity prophylaxis.

All articles or patents mentioned herein are hereby incorporated byreference. The following examples illustrate various aspects of theinvention but are in no way intended to limit the scope thereof.

EXAMPLES Materials and Methods

Cells

A subclone of the FRhK-4 cell line, 11-1, was used in these studiesbecause growth of HAV in this cloned cell line is more efficient than inthe parent cell line (S. U. Emerson, unpublished data). Cells weremaintained in Dulbecco's modified Eagle medium (DMEM) supplemented with10% fetal calf serum, glutamine, non-essential amino acids, 50 μg/mlgentamycin sulfate and 2.5 μg/ml amphotericin B (Fungizone) (10% DMEM).

Reverse Transcription, Polymerase Chain Reaction and DNA Sequencing

An AGM-27 virus stock consisting of 10% (w/v) liver homogenate inphosphate-buffered saline (pH 7.2) (Tsarev, A. A., et al. (1991) J. Gen.Virol., 72:1677-1683) was the source of viral RNA for cloning of cDNAfragments generated by reverse transcription-polymerase chain reaction(RT-PCR). Briefly, RNA was isolated from 5-10 μl of liver homogenate byeither the guanidinium isothiocyanate extraction procedure (Chomczymski,P. et al. (1987) Anal. Biochem., 162: 156-159) or with Trizol reagent(Gibco-BRL, Bethesda, Md.) following the manufacturer's instructions.Glycogen (20 μg; Boehringer Mannheim, Indianapolis, Ind.) was added as acarrier prior to precipitation with isopropanol. The RNA was resuspendedin 10 μl sterile water to which 1 μl of a 10 μM stock of reverse primerwas added. The solution was heated at 65° C. for 3 minutes and cooled atroom temperature for 5 minutes to facilitate primer binding to thetemplate. The reverse transcription reaction was performed in a finalvolume of 20 μl in a reaction mixture consisting of 10 mM Tris-HCl (pH8.4), 50 mM KCl, 2.5 mM MgCl₂, 40 U RNasin (Promega Biotech, Madison,Wis.), 1 mM of each deoxynucleoside triphosphate (dNTP) and 8 U avianmyeloblastosis virus reverse transcriptase. After synthesis of cDNA at42° C. for 60 minutes, PCR amplification was performed in a total volumeof 100 μl of 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM ofeach dNTP, 4 U Taq polymerase (Perkin-Elmer Corp, Norwalk, CT) and 0.5mM each of forward and reverse primers. When necessary, silent mutationswere incorporated into the primers to generate restriction enzyme sitesrequired for subsequent cloning steps. PCR reaction consisted of 35cycles of 1 minute of incubation at 94° C., 1 minute of incubation at45° C. and 1-3 minutes of incubation at 72° C. followed by a singlecycle at 72° C. for 10 minutes. A second round of PCR using nestedprimers was performed if necessary. The PCR products were purified fromlow melting agarose gels by phenol extraction or with a gel purificationkit (Qiagen, Chatsworth, Calif.).

All PCR generated fragments or clones containing PCR generated DNA weresequenced using either Sequence (United States Biochemical Corp.,Cleveland, Ohio) following the manufacturer's instructions or theApplied Biosystems 373A automated DNA sequencer using a modified Sangermethod.

cDNA Clones

All nucleotide number assignments herein are based on the genomic map ofwild-type HM-175 shown in FIG. 12 (which corresponds to FIG. 1 of Cohenet al. (1987) J. Virol., 61:50-59). The pHAV/7 plasmid (Cohen, J. I., etal. (1987) Proc. Natl. Acad. Sci., 84:2497-2501, the pHAV/7 plasmid wasdeposited with the American Type Culture Collection (ATCC) on August 7,1987 and has ATCC accession number 67495) was modified byoligonucleotide-directed mutagenesis to include a PpuM1 site atnucleotide (nt) 3987-3993 (plasmid 32Y; S. U. Emerson and Y. K. Huang,unpublished results). In addition, HAV/7 has a naturally occurring EcoRIsite at nucleotides 4977-4982 near the 3′ end of the 2C gene. As thissite was not present in the AGM-27 consensus sequence, an EcoRI site wasengineered at the analogous position in the AGM-27 gene (T→C mutation atAGM nt 4982) by primer-directed mutagenesis using PCR. To facilitatecloning of intragenic chimeras in 2C, 32Y was further mutagenized usingPCR (AUG mutation at nt 4358) to include an AflII site at nt 4353-4358(plasmid pGRl). A natural AflII site is present at this position in theAGM-27 consensus sequence. The pGR2 chimera was generated by cloning thePpuM1-EcoRI fragment of the AGM-27 consensus sequence (AGM-27 genomicsequence is disclosed in SEQ. ID. NO:1 in U.S. Pat. No. 5,476,658; theAGM-27 virus was deposited with the ATCC on August 24, 1992 and has ATCCaccession number VR 2380) (nt 3996-4981 of AGM-27 2C gene, which encodesamino acids 1-328 of the 2C gene) into the HAV/7 background of p32Y. The2C gene of pGR2 had three nucleotide differences from the AGM-27consensus sequence at positions 4211 (C to T transition), 4280 (G to Atransition) and 4397 (T to C transition) but none changed the amino acidsequence. Because the EcoR1 site at nt 4977-4982 was used for cloning,the pGR2 plasmid contained a glutamic acid residue which is present inHAV/7 at amino acid position 331 in 2C instead of a lysine residue whichis present in the AGM-27 consensus sequence. Thus, of the 31 amino aciddifferences between the 2C protein of AGM-27 and HAV/7, 30 are presentin GR2.

The chimera pGR3 was generated by cloning the PpuM1-AflII fragment fromplasmid pGR2 (AGM-27 2C nt sequence 3996-4357, which encodes amino acids1-121 of the 2C gene) into pGR1. The chimera pGR4 was generated bycloning the AflII-EcoR1 fragment from plasmid pGR2 (AGM-27 2C ntsequence 4354-4981, which encodes amino acids 120-328 of the 2C gene)into pGR1. 17 and 13 of the 31 amino acid differences between the 2Cproteins of AGM-27 and HAV/7 are present in GR3 and GR4, respectively.

Similarly, the PpuM1-AflII or AflII-EcoR1 segments of pGR2 were replacedwith wild-type HM-175 sequences to generate clones pGR14 and pGR13,respectively (FIG. 1). The pGR15 clone contained AGM-27 sequence of 2Bfrom nt 3758-3988. The pGR9 plasmid contained the truncated AGM-27 2Bsequence and 2C sequences from nt 3758-4981 in the HAV/7 background(FIG. 1).

Establishment of Virus Stocks

All full-length cDNAs were cloned in PGEM1 (Promega Biotech). In vitrotranscription and transfection assays were performed as describedpreviously by Emerson et al. (Emerson, S. U., et al. (1991) J. Virol.,65:4882-4886) with a few modifications. Briefly, Sp6 polymerase (PromegaBiotech) transcripts synthesized from 5 μg of DNA linearized with HaeIIwere transfected without purification into 11-1 cells. Transfection ofcells was accomplished by the DEAE-dextran method. One week aftertransfection, half of the cells in each flask were passaged tocoverslips and the fraction of cells which contained viral antigen wasestimated by immunofluorescence microscopy (Emerson, S. U., et al.(1991) J. Virol., 65:4882-4886). Transfected cells were harvested bytrypsinization when >80% of the cells were infected. Viruses werereleased from the harvested cells by at least three cycles offreeze-thawing to generate the working virus stocks.

Radioimmunofocus Assay (RIFA)

RIFA was used to determine focus size of different chimeric viruses andto quantify virus titers. RIFA was a modification of that described byLemon et al. (1983) (J. Clin. Microbiol., 17:834-839) and Anderson etal. (1987) (“Positive Strand RNA Viruses” MA Brinton and RR Reichert(ed.) AR Liss Inc. NY) and was essentially performed as previouslydescribed (Funkhouser, A. W., et al. (1994) J. Virol., 68:148-157).Cells (11-1) were grown on Thermolux round 25 mm coverslips fixed to thebottom of each well in 6-well plates. Virus was adsorbed to cells for2-4 hours at 34.5° C. in a CO₂ incubator.

Infected cells were overlaid with 5 ml of 0.5% agarose medium andincubated at 34.5° C. in a CO₂ incubator for 10 days. Cells were fixedwith acetone and were either processed immediately or stored at -20° C.Viral antigen was detected with a primary antibody consisting ofchimpanzee hyperimmune serum (S. U. Emerson, unpublished results) and asecondary antibody of ¹²⁵I-labelled sheep anti-human IgG F(ab′)₂fragment (Amersham Corporation, Arlington Heights, Ill.). Foci werevisualized by autoradiography.

Growth Curve Assays

Growth curve assays were performed to evaluate the relative rates ofreplication of viruses in 11-1 cells. Cell monolayers which were >80%confluent in 96-well plates (Falcon) were infected with 0.2 ml of virusdiluted in 10% DMEM at a multiplicity of infection of 6radioimmunofocus-forming units (RFU) per cell. Cells were incubated withvirus for 2-4 hours at 34.5°C. in a CO₂ incubator after which the cellswere washed 4 times with 10DMEM and overlaid with 0.2 ml 10% DMEM. Atvarious times, the medium overlaying the cells, which contained virusreleased from cells, was removed and saved and the volume was adjustedto 0.3 ml with 10i DMEM. The adherent cells in the wells were washedonce with 0.2 ml trypsin solution at room temperature and weresubsequently incubated with 0.1 ml trypsin solution at 34.5° C. untilthe cells exhibited a rounded morphology. The trypsinized cells werethen harvested by vigorous pipetting and combined with the medium thathad been previously removed. The total virus sample (0.4 ml) was storedat −80° C. Cells were lysed by at least three cycles of freeze-thawingand virus was quantified either by slot blot hybridization or by RIFA.Either HAV/7 or a modified clone of HAV/7 containing a silent mutationwas used as a standard in these experiments to measure growth of thefully cell culture-adapted virus. Since there was no significantdifference in the growth properties of these two viruses, they were usedinterchangeably and for simplicity, are always referred to as HAV/7 inthis study.

Slot Blot Hybridization

RNA was isolated from growth curve samples using Trizol reagent(Gibco-BRL) according to the manufacturer's instructions with thefollowing modifications: A 130 μl aliquot of sample was extracted withan equal volume of Trizol. After the 15 minute centrifugation step toseparate the aqueous and organic phases, 100 μl of the aqueous phase wasremoved and added to 320 μl of a 1:1 solution of 10×SSC (1×SSC is 0.15 MNaCl and 0.015 M sodium citrate (pH 7.0)) and formaldehyde. Viral RNAwas quantified by slot blot analysis using a negative strand ³²p-labelled RNA probe spanning the complete HAV/7 genome. Hybridization wasat 50° C. for at least 16 hours. Blots were washed three times for 30minutes each with 2×SSPE (1×SSPE is 10 mM sodium phosphate, 0.18 M NaCl,1 mM EDTA (pH 7.4)) with 0.1% sodium dodecyl sulphate (SDS) at roomtemperature and once with 0.1×SSPE, 0.1% SDS at 64° C. for one hour.Autoradiography was performed and a Deskscan II scanner (HewlettPackard) with NIH Image analysis package (Wayne Rasband, public domainsoftware, Bethesda, Md.) was used to quantify viral RNA from each timepoint in the growth curve. At least two or more sister clones wereassayed for each virus construct.

Virulence Studies in Tamarins

The ability of AGM-27 2C chimeras GR2, GR3 and GR4 to cause disease intamarins was evaluated. Each of two tamarins for each chimera wasinoculated intravenously with approximately 10^(3.8) tissue cultureinfectious dose equivalents of the GR2 virus in a 0.5 ml volume ofinoculum or with approximately 10^(4.8) tissue culture infectious doseequivalents of the GR3 or GR4 viruses in a 0.5 ml volume of inoculum.The estimated number of genome equivalents in the inoculum wasdetermined by RIFA. Blood samples were collected and needle liverbiopsies were performed weekly on each animal for at least 2 weeksbefore and 16 weeks after inoculation with virus. The blood samples wereanalyzed for seroconversion to anti-HAV with a commercial assay (AbbottLaboratories, North Chicago, Ill.) and for serum alanine aminotransferase (ALT) and isocitrate dehydrogenase (ICD) levels withstandard techniques (Metpath, Rockville, Md.). Histopathology wasdetermined under code and scored on a scale of 1 to 4 depending on theseverity of the hepatitis; 1 corresponded to mild hepatitis and 4 tosevere hepatitis.

Stool samples from the GR2, GR3 or GR4-inoculated tamarins were alsoanalyzed for the presence of excreted virus by RT-PCR. Briefly, a 10%(w/v) suspension of stool in 10 mM Tris (pH 7.0) and 0.135 M NaCl wasprepared in a and clarified by low-speed centrifugation to remove largeparticulate matter. Viral RNA was extracted from a 100 μl aliquot ofclarified sample with 1 ml of Trizol reagent (Gibco-BRL) following themanufacturer's instructions and RT-PCR was performed to amplify aspecific region of the viral genome. The amplified DNA was purified witha PCR fragment purification kit (Qiagen) and was sequenced to determinethe identity of the excreted virus.

Virulence Studies In Chimpanzees

Two chimpanzees each were inoculated intravenously with approximately10⁵ tissue culture infectious dose equivalents of either the GR4 or GR2virus in a 0.5 ml volume of inoculum. Blood samples were collected andneedle liver biopsies were performed weekly on each animal for at least2 weeks before and 16 weeks after inoculation with virus. The bloodsamples were analyzed for seroconversion to anti-HAV with commercialassay and for serum alanine amino transferase (ALT) and isocitratedehydrogenase (ICD) levels. Histopathology was determined under code andscored on a scale of 1 to 4 as for the tamarins.

The housing, maintenance, and care of all animals met or exceeded allrequirements for primate husbandry.

Example 1 Construction of Chimeras between AGM-27 and HM-175

The 2B and 2C gene products are the most important determinants ofefficient growth of HAV in cell culture (Emerson, S. U., et al. (1992)J. Virol., 66:650-654). Chimeras containing the AGM-27 truncated 2Band/or 2C gene in the background of the cell culture-adapted HM-175virus genome were constructed as described in the methods section andare shown schematically in FIG. 1. The 2C gene was further subdividedand intragenic chimeras between the simian and human 2C genes weregenerated (FIG. 1, GR3 and GR4). The amino acid sequence differencesbetween the HM-175 (for either wide-type or HAV/7) and AGM-27 2Cproteins are clustered at the amino-terminus and carboxy-terminus of theprotein (FIG. 2). The intragenic 2C chimeras were constructed in orderto evaluate the effect of separating the two clusters of amino acidresidues at the ends of the 2C protein, which differ between HM-175 andAGM-27, from each other. Results obtained with these chimeras aredescribed in the following Examples.

Example 2 RIFA of HAV/7, AGM-27 and the GR2 Chimera

The radioimmunofocus assay is one method which can be used to evaluatethe relative growth properties of viruses. The cell culture-adaptedHM-175 virus (HAV/7) grew well in cell culture and formed large foci(FIG. 3). AGM-27, which is a wild-type virus, clearly grew in cellculture but had a small focus phenotype (FIG. 3). By comparison,wild-type HM-175 grew so poorly that visible foci were not detected bythis assay (data not shown). Replacement of the HAV/7 2C gene withAGM-27 sequences (chimera GR2) drastically reduced the ability of thevirus to grow (FIG. 3). The only differences between HAV/7, which formedlarge foci, and the chimera GR2, which had a small focus phenotype, arein the 2C gene, demonstrating the significant contribution of thesequences in 2C to growth of HAV in cell culture.

Example 3 RIFA of HAV/7 and Chimeras GR2, GR3, GR4. GR15 and GR9

Replacement of the 2C gene of HAV/7 with AGM-27 sequences drasticallyreduced the size of the foci that were formed (FIG. 4B). Thesimian-human intragenic 2C chimeras GR3 and GR4 formed intermediatesized foci (FIGS. 4C and 4D). Replacement of the HAV/7 truncated 2B genewith AGM-27 sequences decreased the size of the foci only slightly, ifat all (FIG. 4E). However, a chimera containing both the AGM-27truncated 2B gene and 2C gene in the HAV/7 background formed foci thatwere even smaller (FIG. 4F) than those formed by the chimera containingonly the AGM-27 2C gene, again demonstrating the negative influence ofthe AGM-27 truncated 2B sequence in the context of the homologous 2C.

Example 4 Kinetic Studies Showing Relative Growth Rates of HAV/7 and theChimeras

Kinetic studies were conducted as a quantitative measure of the relativegrowth efficiencies of HAV/7 and the chimeric viruses. Cells wereinfected with a high multiplicity of virus (6 radioimmunofocus units(RFU)/cell) to ensure that almost every cell was infected. Increase invirus titer over time was therefore a measure of virus replication. RNAwas extracted from samples harvested at each time point and quantifiedby slot blot hybridization and densitometry. Comparison of growth ofHAV/7 with a chimera containing the AGM-27 2C gene in this samebackground (GR2) shows that consistent with the small focus phenotypefor GR2 shown previously (FIG. 3) the AGM-27 2C sequences greatlyreduced the ability of the virus to grow (FIG. 5). Substitution of thecell culture-adapted 2C gene with that from the wild-type human virusalso reduced the growth potential of the virus (36Y) although not asdrastically as that observed with the simian-human chimera (FIG. 5). Therelative rates of growth of HAV/7 and the 2C chimeras as measured byRIFA were similar to that observed by slot blot analysis (data notshown). Both assays were performed for most chimeras studied and thereconsistently was good correlation between the results obtained by thetwo assays.

Kinetic assays were also performed to evaluate the ability of theintragenic 2C chimeras to grow in cell culture. Quantitation was againby slot blot hybridization and densitometry. Chimeras containing AGM-27sequences in either half of 2C (GR3 and GR4) had an intermediate growthphenotype (FIG. 6) as they grew less well than HAV/7 but better than thechimera containing the entire AGM-27 2C sequence (GR2). These datademonstrate that the clusters of amino acid residues that are unique toAGM-27 at either end of the 2C protein have a negative effect on growthand that this effect is additive. Growth of the GR4 chimera wasespecially sensitive to the status of the cells and GR4 grew at a rateslightly greater or less than that of the GR3 chimera in differentexperiments. In every case however, it grew significantly less well thandid HAV/7. Kinetic studies were also performed with chimeras containingwild-type HM-175 sequence in one half and AGM-27 sequences in the otherhalf of 2C in the HAV/7 background (GR13 and GR14; FIG. 1). Thesechimeras grew but not as well as the analogous chimeras containing HAV/7sequences instead of wild-type HM-175 sequence (data not shown), againemphasizing the importance of the mutations in 2C that were acquiredduring passage of HM-175 in cell culture.

The truncated AGM-27 2B gene by itself in the HAV/7 background (GR15)had only a minor effect, if any, on virus growth (FIG. 7). However inconjunction with the homologous 2C (GR9), the truncated AGM-27 2B geneconsistently had a significant negative effect on virus growth (FIG. 7).The results of the kinetic studies presented in FIGS. 5-7 wereconsistent with the relative sizes of the foci observed for thedifferent chimeric viruses. Moreover, the RIFA and kinetics studiesdemonstrated that the 2C gene is a major determinant of the efficiencyof growth in cell culture and that the AGM-27 2C gene significantlydecreases the ability of HAV/7 to grow in cell culture. In addition, thefact that the intragenic chimeras GR3 and GR4 are viable and grow atintermediate levels suggests that each cluster of sequence differencespresent at the ends of the 2C gene in AGM-27 has a negative effect ongrowth.

Example 5 Virulence Studies in Tamarins of the Chimeras GR2, GR3 and GR4

AGM-27 is virulent in tamarins but attenuated in chimpanzees (Emerson,S. U., et al. (1996) J. Infect. Dis., 173:592-597) while HAV/7 has anattenuated phenotype in tamarins (Cohen, J. I., et al. (1989) J. Virol.,63:5364-5370). The GR2, GR3 and GR4 chimeras were each inoculated intotwo tamarins intravenously and serum liver enzyme levels, antibodytiters and liver pathology were evaluated. The pattern of changes in ICDand ALT levels during the course of infection of all animals weresimilar.

The results presented in FIGS. 8A and 8B show that the GR2 chimera wasvirulent in tamarins, causing significant increases in serum liverenzyme levels and seroconversion for HAV by week 5 after inoculation.Liver histology showed mild (1+) to moderate (2+) hepatitis 6-9 weeksafter inoculation in one animal (FIG. 8A) and mild (1+) to moderatelysevere (3+) hepatitis 5-8 weeks after inoculation in the second animal(FIG. 8B). Virus specific RT-PCR amplification of fecal samples fromweeks 5, 6 and 7 were positive either after one round (weeks 5 and 6) orafter two rounds (week 7) of PCR with nested primers. A significantamount of virus was therefore being excreted concurrent with the peakelevation of liver enzyme values in the serum and seroconversion in theanimals. Partial sequence analysis of virus genomes amplified from thestool samples showed that the sequence of the excreted virus was thesame as that of the chimeric virus in the inoculum. These resultsdemonstrate that the 2C gene of AGM-27 can confer the phenotype ofvirulence for tamarins to an otherwise attenuated virus (HAV/7).

Tamarins inoculated with a chimera containing AGM-27 sequence in the 5′half of the 2C gene in the HAV/7 background (GR3) showed no significantincrease in serum liver enzyme levels in either animal (FIGS. 9A and9B). Seroconversion occurred 7 weeks after inoculation in one animal(FIG. 9A) but the level of anti-HAV antibodies was relatively low. Therewas seroconversion in the second animal in week 15. (FIG. 9B).

Tamarins inoculated with a chimera containing AGM-27 sequence in the 3′half of the 2C gene in the HAV/7 background (GR4) seroconverted at 6weeks after inoculation (FIGS. 10A and 10B). The serum liver enzymelevel was slightly elevated in one animal (FIG. 10A) and showed nosignificant increase in the second animal (FIG. 10B).

The data from these tamarin studies suggest that the 2C gene plays acritical role in virulence of HAV in tamarins and that the AGM-27sequence at the 3′ half of the 2C gene appears to make a greatercontribution to this phenotype than the AGM-27 sequence at the 5′ halfof the 2C gene.

Example 6 Virulence of the GR2 and GR4 Chimeras in Chimpanzees

Chimpanzees inoculated with the GR4 chimera seroconverted either 8 or 15weeks after inoculation (FIGS. 11A-11D) and chimpanzees inoculated withthe GR2 chimera seroconverted either 7 or 17 weeks after inoculation(FIGS. 12A and 12B). None of the animals had any increase in serum liverenzyme levels (FIGS. 11A-11D, 12A and 12B).

Example 7 Summary of The Data From the Tamarins and ChimpanzeesInoculated With the HAV/7/AGM-27 Chimeras

The data from the tamarin and chimpanzee studies are summarized in Table1.

TABLE 1 Peak Peak Seroconversion ICD ICD Anti-Hav Tamarin Virus (week)(week) value titer 782 GR2 5 6 5190(1)* 1:16000 783 GR2 5 6 7520(2)*1:32000 808 GR3 7 none baseline 1:200 790 GR3 15  none baseline 1:1600799 GR4 6 7 1236(2)* 1:8000 818 GR4 6 none baseline 1:1600 Peak PeakPeak Seroconversion ALT ALT Anti-Hav Chimpanzee Virus (week) (week)value titer 1545 GR2  8 none baseline 1:200 1547 GR2 17 none baseline1:40 1558 GR4 10 none baseline 1:800 1564 GR4  8 none baseline 1:40 *Thenumber within the brackets refers to the number of weeks that the serumICD level was above the baseline value.

The GR2 and GR4 chimeras thus appear to be attenuated in both tamarinsand chimpanzees. These results suggest that the GR2 and GR4 chimeras mayfunction as live attenuated vaccines to offer protection againstchallenge with human virulent HAV.

Example 8 Challenge of Chimpanzees Inoculated With AGM-27/HAV/7 Chimera

Chimpanzees are inoculated with 10⁵ tissue culture infectious doseequivalents of GR4 virus either by the oral, intramuscular, intradermalor intravenous route of infection. Blood samples are collected andneedle liver biopsies are performed weekly on each animal for at leasttwo weeks before inoculation and for the duration of the study. Theblood samples are analyzed for seroconversion to anti-HAV by acommercial assay (Abbott Laboratories, North Chicago Ill.) and for serumalanine amino transferase (ALT) and isocitrate dehydrogenase (ICD)levels with standard techniques. Liver tissue will also be examined forsigns of hepatitis. Three months after antibodies are first detected inserum using a commercial assay, the animals are challenged with 10³ to10 ⁴ chimpanzee dose equivalents of virulent HAV (HM-175 or SD-11).After challenge with the virulent strain of HAV, the animals areprotected as measured by biochemical (ALT or ICD), serological (levelsof anti-HAV antibodies) and histopathological analyses of the animalsfor several months after challenge.

3 7493 base pairs nucleic acid single linear not provided 1 TTCAAGAGGGGTCTCCGGGA ATTTCCGGAG TCCCTCTTGG 40 AAGTCCATGG TGAGGGGACT TGATACCTCACCGCCGTTTG 80 CCTAGGCTAT AGGCTAAATT TTCCCTTTCC CTTTTCCCTT 120 TCCTATTCCCTTTGTTTTGC TTGTAAATAT TAATTCCTGC 160 AGGTTCAGGG TTCTTAAATC TGTTTCTCTATAAGAACACT 200 CATTTTTCAC GCTTTCTGTC TTCTTTCTTC CAGGGCTCTC 240CCCTTGCCCT AGGCTCTGGC CGTTGCGCCC GGCGGGGTCA 280 ACTCCATGAT TAGCATGGAGCTGTAGGAGT CTAAATTGGG 320 GACACAGATG TTTGGAACGT CACCTTGCAG TGTTAACTTG360 GCTTTCATGA ATCTCTTTGA TCTTCCACAA GGGGTAGGCT 400 ACGGGTGAAACCTCTTAGGC TAATACTTCT ATGAAGAGAT 440 GCCTTGGATA GGGTAACAGC GGCGGATATTGGTGAGTTGT 480 TAAGACAAAA ACCATTCAAC GCCGGAGGAC TGACTCTCAT 520CCAGTGGATG CATTGAGTGG ATTGACTGTC AGGGCTGTCT 560 TTAGGCTTAA TTCCAGACCTCTCTGTGCTT AGGGCAAACA 600 TCATTTGGCC TTAAATGGGA TTCTGTGAGA GGGGATCCCT640 CCATTGACAG CTGGACTGTT CTTTGGGGCC TTATGTGGTG 680 TTTGCCTCTGAGGTACTCAG GGGCATTTAG GTTTTTCCTC 720 ATTCTTAAAT AATAATGAAC ATGTCTAGACAAGGTATTTT 760 CCAGACTGTT GGGAGTGGTC TTGACCACAT CCTGTCTTTG 800GCAGACATTG AGGAAGAGCA AATGATTCAA TCAGTTGATA 840 GGACTGCAGT GACTGGTGCTTCTTATTTTA CTTCTGTGGA 880 TCAATCTTCA GTTCATACAG CTGAGGTTGG ATCACACCAG920 GTTGAACCTT TGAGAACCTC TGTTGATAAA CCCGGTTCAA 960 AGAAGACTCAGGGAGAGAAA TTTTTCTTGA TTCATTCTGC 1000 AGATTGGCTT ACTACACATG CTCTTTTCCATGAAGTTGCA 1040 AAATTGGATG TGGTGAAATT ATTATACAAT GAGCAGTTTG 1080CTGTTCAAGG GTTGTTGAGA TACCATACAT ATGCAAGATT 1120 TGGCATTGAA ATTCAAGTTCAGATAAACCC TACACCTTTC 1160 CAACAGGGGG GATTGATCTG TGCTATGGTT CCTGGTGACC1200 AGAGCTATGG TTCTATAGCA TCATTGACTG TTTATCCTCA 1240 TGGTTTGTTAAATTGCAATA TTAACAATGT GGTTAGAATA 1280 AAGGTTCCAT TTATTTACAC AAGAGGTGCTTACCACTTTA 1320 AAGATCCACA ATACCCAGTT TGGGAATTGA CAATTAGAGT 1360TTGGTCAGAA TTAAATATTG GGACAGGAAC TTCAGCTTAT 1400 ACTTCACTCA ATGTTTTAGCTAGATTTACA GATTTGGAGT 1440 TGCATGGATT AACTCCTCTT TCTACACAAA TGATGAGAAA1480 TGAATTTAGG GTCAGTACTA CTGAGAATGT GGTGAATCTG 1520 TCAAATTATGAAGATGCAAG AGCAAAGATG TCTTTTGCTT 1560 TGGATCAGGA AGATTGGAAA TCTGATCCGTCCCAGGGTGG 1600 TGGGATCAAA ATTACTCATT TTACTACTTG GACATCTATT 1640CCAACTTTGG CTGCTCAGTT TCCATTTAAT GCTTCAGACT 1680 CAGTTGGTCA ACAAATTAAAGTTATTCCAG TTGACCCATA 1720 TTTTTTCCAA ATGACAAATA CGAATCCTGA CCAAAAATGT1760 ATAACTGCTT TGGCTTCTAT TTGTCAGATG TTTTGTTTTT 1800 GGAGAGGAGATCTTGTCTTT GATTTTCAAG TTTTTCCCAC 1840 CAAATATCAT TCAGGTAGAT TACTGTTTTGTTTTGTTCCT 1880 GGCAATGAGC TAATAGATGT TTCTGGAATC ACATTAAAGC 1920AAGCAACTAC TGCTCCTTGT GCAGTAATGG ATATTACAGG 1960 AGTGCAGTCA ACTTTGAGATTTCGTGTTCC CTGGATTTCT 2000 GACACTCCTT ACAGAGTGAA CAGGTATACA AAGTCAGCAC2040 ATCAGAAAGG TGAGTACACT GCCATTGGGA AGCTTATTGT 2080 GTATTGTTATAACAGATTGA CCTCTCCTTC TAACGTTGCT 2120 TCCCATGTCA GAGTGAATGT TTATCTTTCAGCAATTAACT 2160 TGGAATGTTT TGCTCCTCTT TATCATGCTA TGGATGTTAC 2200TACACAAGTT GGAGATGATT CTGGAGGTTT TTCAACAACA 2240 GTTTCTACAG AACAGAATGTTCCAGATCCC CAAGTTGGTA 2280 TAACAACCAT GAAAGATTTG AAAGGAAAAG CTAACAGAGG2320 GAAAATGGAT GTTTCAGGAG TACAAGCACC TGTGGGAGCT 2360 ATCACAACAATTGAGGATCC AGTTTTAGCA AAGAAAGTAC 2400 CTGAGACATT TCCTGAATTG AAACCTGGAGAATCCAGACA 2440 TACATCAGAT CATATGTCCA TCTACAAGTT TATGGGAAGG 2480TCTCATTTCT TGTGCACTTT TACATTCAAT TCAAATAATA 2520 AAGAGTACAC ATTTCCTATAACCTTGTCTT CAACCTCTAA 2560 TCCTCCTCAT GGTTTGCCAT CAACACTGAG GTGGTTTTTC2600 AACTTGTTTC AGTTGTATAG AGGGCCTTTA GATCTGACAA 2640 TTATTATTACAGGAGCAACT GATGTAGATG GCATGGCCTG 2680 GTTCACTCCA GTAGGTCTTG CCGTTGATACTCCTTGGGTA 2720 GAGAAGGAGT CAGCTTTGTC TATTGACTAC AAAACTGCTC 2760TTGGAGCTGT CAGATTTAAC ACAAGGAGAA CAGGGAACAT 2800 TCAGATTAGA TTACCATGGTATTCTTATTT ATATGCTGTG 2840 TCTGGAGCAC TGGATGGTTT GGGTGACAAG ACAGATTCTA2880 CATTTGGATT GGTTTCTATT CAGATTGCAA ATTACAATCA 2920 TTCTGATGAATACTTGTCTT TTAGTTGTTA TTTGTCTGTC 2960 ACAGAACAAT CAGAGTTTTA TTTTCCCAGAGCTCCATTGA 3000 ACTCAAATGC CATGTTATCC ACTGAATCAA TGATGAGCAG 3040AATTGCAGCT GGAGACTTGG AGTCATCAGT GGATGATCCT 3080 AGATCAGAGG AAGATAAAAGATTTGAGAGT CATATAGAAT 3120 GCAGGAAGCC ATATAAAGAA CTGAGATTAG AAGTTGGGAA3160 ACAAAGACTC AAGTATGCTC AGGAAGAATT GTCAAATGAA 3200 GTACTTCCACCCCCTAGGAA AATGAAGGGA CTGTTTTCAC 3240 AAGCCAAAAT TTCTCTTTTT TATACTGAGGAGCATGAAAT 3280 AATGAAGTTT TCCTGGAGAG GTGTGACTGC TGATACTAGA 3320GCTTTAAGGA GGTTTGGATT CTCTTTGGCC GCAGGCAGAA 3360 GTGTGTGGAC TCTTGAAATGGATGCTGGGG TTCTTACTGG 3400 GAGACTGATT AGATTGAATG ATGAGAAATG GACAGAAATG3440 AAGGATGACA AGATTGTTTC ATTGATTGAA AAGTTTACAA 3480 GTAACAAATATTGGTCCAAA GTGAATTTCC CACATGGGAT 3520 GTTGGATCTT GAAGAAATTG CTGCCAATTCTAAGGATTTT 3560 CCTAACATGT CTGAAACGGA TTTGTGTTTC TTGCTGCATT 3600GGTTAAATCC AAAGAAAATT AATTTAGCAG ATAGAATGCT 3640 TGGATTGTCT GGAGTTCAGGAAATTAAAGA ACAAGGTGTT 3680 GGATTAATAG CAGAGTGTAG AACTTTCTTA GATTCTATTG3720 CTGGAACTTT AAAATCTATG ATGTTTGGAT TTCATCATTC 3760 TGTGACTGTTGAAATTATAA ACACTGTGCT CTGTTTTGTT 3800 AAGAGTGGAA TTTTGCTTTA TGTAATACAACAATTGAATC 3840 AGGATGAACA TTCTCACATA ATTGGTTTGT TGAGAGTCAT 3880GAATTATGCA GATATTGGTT GTTCAGTTAT TTCATGTGGC 3920 AAAGTTTTTT CCAAAATGCTGGAAACAGTC TTTAATTGGC 3960 AAATGGACTC CAGAATGATG GAGTTAAGGA CTCAGAGTTT4000 TTCCAACTGG TTAAGAGATA TTTGTTCTGG GATCACCATT 4040 TTTAAAAACTTCAAGGATGC AATTTATTGG CTTTATACAA 4080 AATTAAAGGA CTTTTATGAA GTGAATTATGGCAAGAAGAA 4120 GGACATTTTA AATATTCTTA AAGATAACCA ACAAAAAATA 4160GAGAAAGCCA TTGAGGAAGC CGATGAATTT TGCATTTTGC 4200 AAATCCAAGA TGTGGAAAAATTTGAACAGT ATCAGAAAGG 4240 GGTTGACTTG ATACAAAAAT TGAGAACTGT TCATTCAATG4280 GCTCAGGTTG ATCCAAATTT AATGGTTCAT TTGTCACCTT 4320 TGAGAGATTGTATAGCAAGA GTTCATCAGA AACTTAAAAA 4360 CCTTGGATCT ATAAATCAGG CAATGGTAACGAGATGTGAG 4400 CCAGTTGTTT GTTATTTATA TGGCAAAAGA GGGGGAGGAA 4440AGAGCTTAAC ATCAATTGCA TTGGCAACCA AAATTTGTAA 4480 ACATTATGGT GTTGAGCCTGAAAAGAATAT CTATACTAAA 4520 CCTGTGGCTT CAGATTACTG GGATGGATAT AGTGGACAAT4560 TAGTTTGCAT CATTGATGAT ATTGGCCAAA ACACAACAGA 4600 TGAGGATTGGTCAGATTTTT GTCAGTTAGT GTCAGGATGT 4640 CCAATGAGAT TAAACATGGC CTCTCTTGAGGAGAAGGGTA 4680 GGCATTTTTC TTCTCCTTTT ATAATAGCAA CTTCAAATTG 4720GTCAAATCCA AGTCCAAAAA CAGTTTATGT TAAGGAAGCA 4760 ATTGACCGCA GACTCCATTTCAAGGTTGAA GTTAAACCTG 4800 CTTCATTTTT CAAAAATCCT CACAATGATA TGTTGAATGT4840 TAATTTAGCT AAAACAAATG ATGCAATCAA AGATATGTCT 4880 TGTGTTGATTTGATAATGGA TGGACATAAT GTTTCATTGA 4920 TGGATTTGCT CAGTTCTTTA GTCATGACAGTTGAAATTAG 4960 AAAACAAAAC ATGACTGAAT TCATGGAGTT GTGGTCTCAG 5000GGAATTTCAG ATGATGATAA TGATAGTGCA GTAGCTGAGT 5040 TTTTCCAGTC TTTTCCATCTGGTGAACCAT CGAACTCTAA 5080 ATTATCTGGC TTTTTCCAAT CTGTTACTAA TCACAAGTGG5120 GTTGCTGTGG GAGCTGCAGT TGGCATTCTT GGAGTGCTCG 5160 TTGGAGGATGGTTTGTGTAT AAGCATTTCT CCCGCAAAGA 5200 GGAGGAACCA ATCCCAGCTG AAGGGGTATATCATGGTGTA 5240 ACTAAGCCCA AGCAAGTGAT TAAATTAGAT GCAGATCCAG 5280TAGAATCTCA GTCAACTTTG GAAATAGCAG GACTGGTTAG 5320 GAAGAACTTG GTTCAGTTTGGAGTTGGAGA GAAGAATGGA 5360 TGTGTGAGAT GGGTTATGAA TGCCTTGGGA GTGAAAGATG5400 ATTGGCTGCT TGTGCCTTCC CATGCTTATA AATTTGAGAA 5440 AGATTATGAAATGATGGAGT TTTATTTTAA TAGAGGTGGA 5480 ACTTACTATT CAATTTCAGC TGGTAATGTTGTTATTCAAT 5520 CTTTGGATGT GGGATTCCAG GATGTTGTTC TGATGAAGGT 5560TCCTACAATT CCTAAGTTTA GAGATATTAC TCAGCATTTT 5600 ATTAAGAAAG GGGATGTGCCTAGAGCTTTG AATCGCCTGG 5640 CAACATTAGT GACAACTGTA AATGGAACCC CTATGTTAAT5680 TTCTGAGGGC CCACTAAAGA TGGAAGAGAA AGCTACTTAT 5720 GTTCATAAGAAAAATGATGG TACAACAGTT GATTTAACTG 5760 TGGATCAGGC ATGGAGAGGA AAAGGCGAAGGTCTTCCTGG 5800 AATGTGTGGT GGGGCCTTGG TTTCATCGAA TCAATCTATA 5840CAGAATGCAA TCTTGGGCAT CCATGTTGCT GGAGGAAATT 5880 CAATTCTTGT TGCAAAATTGGTTACTCAAG AAATGTTCCA 5920 AAATATTGAT AAGAAAATTG AAAGTCAGAG AATTATGAAA5960 GTGGAGTTTA CTCAGTGTTC AATGAATGTG GTCTCCAAAA 6000 CGCTTTTTAGAAAGAGTCCC ATTTATCATC ACATTGATAA 6040 AACCATGATT AATTTTCCTG CAGCTATGCCCTTTTCTAAA 6080 GCTGAAATTG ATCCAATGGC TGTGATGTTA TCTAAGTATT 6120CATTACCTAT TGTAGAAGAA CCAGAGGATT ATAAAGAGGC 6160 TTCAATTTTT TATCAAAATAAAATAGTGGG TAAGACTCAG 6200 TTAGTTGATG ATTTTTTAGA TCTTGATATG GCCATTACAG6240 GGGCCCCAGG AATTGATGCT ATCAACATGG ATTCATCTCC 6280 TGGATTTCCTTATGTCCAGG AGAAGTTGAC CAAAAGAGAT 6320 TTAATTTGGT TGGATGAAAA TGGTTTATTGCTGGGAGTTC 6360 ATCCAAGATT GGCTCAGAGA ATCTTATTCA ATACTGTCAT 6400GATGGAAAAT TGTTCTGATT TGGATGTTGT TTTTACAACC 6440 TGTCCAAAAG ATGAATTGAGACCATTAGAG AAAGTGTTGG 6480 AATCAAAAAC AAGAGCTATT GATGCTTGTC CTCTGGATTA6520 CTCAATTTTG TGCCGAATGT ATTGGGGTCC AGCTATTAGT 6560 TATTTTCATTTGAATCCAGG TTTCCATACA GGTGTTGCTA 6600 TTGGCATAGA TCCTGATAGA CAGTGGGATGAATTATTTAA 6640 AACAATGATA AGATTCGGAG ATGTTGGTCT TGATTTAGAT 6680TTCTCTGCTT TTGATGCTAG TCTTAGTCCA TTTATGATTA 6720 GAGAAGCAGG TAGAATCATGAGTGAACTAT CTGGAACTCC 6760 ATCCCATTTT GGCACAGCTC TTATCAATAC TATCATTTAT6800 TCCAAGCATT TGCTGTATAA CTGTTGTTAC CATGTCTGTG 6840 GTTCAATGCCCTCTGGGTCT CCTTGTACAG CTTTGCTAAA 6880 TTCAATTATT AATAATGTCA ATTTGTATTATGTGTTTTCC 6920 AAGATATTTG GAAAGTCTCC AGTTTTCTTT TGTCAGGCTT 6960TGAAGATTCT CTGTTATGGA GATGATGTTT TAATAGTTTT 7000 CTCTCGAGAT GTTCAGATTGATAATCTTGA TTTGATTGGA 7040 CAAAAAATTG TAGATGAGTT TAAGAAACTT GGCATGACAG7080 CTACTTCTGC TGACAAGAAT GTACCTCAGC TGAAACCAGT 7120 TTCGGAATTGACTTTTCTCA AAAGATCTTT CAATTTGGTA 7160 GAGGATAGAA TTAGACCTGC AATTTCGGAAAAAACAATTT 7200 GGTCTTTAAT AGCATGGCAG AGAAGTAACG CTGAGTTTGA 7240GCAGAATTTA GAAAATGCTC AGTGGTTTGC TTTTATGCAT 7280 GGCTATGAGT TTTATCAGAAATTTTATTAT TTTGTTCAGT 7320 CCTGTTTGGA GAAAGAGATG ATAGAATACA GACTTAAATC7360 TTATGATTGG TGGAGAATGA GATTTTATGA CCAGTGTTTC 7400 ATTTGTGACCTTTCATGATT TGTTTAAACA AATTTTCTTA 7440 AAATTTCTGA GGTTTGTTTA TTTCTTTTATCAGTAAATAA 7480 AAAAAAAAAA AAA 7493 2227 amino acids amino acid unknownunknown not provided 2 Met Asn Met Ser Arg Gln Gly Ile Phe Gln Thr Val 15 10 Gly Ser Gly Leu Asp His Ile Leu Ser Leu Ala Asp 15 20 Ile Glu GluGlu Gln Met Ile Gln Ser Val Asp Arg 25 30 35 Thr Ala Val Thr Gly Ala SerTyr Phe Thr Ser Val 40 45 Asp Gln Ser Ser Val His Thr Ala Glu Val GlySer 50 55 60 His Gln Val Glu Pro Leu Arg Thr Ser Val Asp Lys 65 70 ProGly Ser Lys Lys Thr Gln Gly Glu Lys Phe Phe 75 80 Leu Ile His Ser AlaAsp Trp Leu Thr Thr His Ala 85 90 95 Leu Phe His Glu Val Ala Lys Leu AspVal Val Lys 100 105 Leu Leu Tyr Asn Glu Gln Phe Ala Val Gln Gly Leu 110115 120 Leu Arg Tyr His Thr Tyr Ala Arg Phe Gly Ile Glu 125 130 Ile GlnVal Gln Ile Asn Pro Thr Pro Phe Gln Gln 135 140 Gly Gly Leu Ile Cys AlaMet Val Pro Gly Asp Gln 145 150 155 Ser Tyr Gly Ser Ile Ala Ser Leu ThrVal Tyr Pro 160 165 His Gly Leu Leu Asn Cys Asn Ile Asn Asn Val Val 170175 180 Arg Ile Lys Val Pro Phe Ile Tyr Thr Arg Gly Ala 185 190 Tyr HisPhe Lys Asp Pro Gln Tyr Pro Val Trp Glu 195 200 Leu Thr Ile Arg Val TrpSer Glu Leu Asn Ile Gly 205 210 215 Thr Gly Thr Ser Ala Tyr Thr Ser LeuAsn Val Leu 220 225 Ala Arg Phe Thr Asp Leu Glu Leu His Gly Leu Thr 230235 240 Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg 245 250 Val SerThr Thr Glu Asn Val Val Asn Leu Ser Asn 255 260 Tyr Glu Asp Ala Arg AlaLys Met Ser Phe Ala Leu 265 270 275 Asp Gln Glu Asp Trp Lys Ser Asp ProSer Gln Gly 280 285 Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr 290295 300 Ser Ile Pro Thr Leu Ala Ala Gln Phe Pro Phe Asn 305 310 Ala SerAsp Ser Val Gly Gln Gln Ile Lys Val Ile 315 320 Pro Val Asp Pro Tyr PhePhe Gln Met Thr Asn Thr 325 330 335 Asn Pro Asp Gln Lys Cys Ile Thr AlaLeu Ala Ser 340 345 Ile Cys Gln Met Phe Cys Phe Trp Arg Gly Asp Leu 350355 360 Val Phe Asp Phe Gln Val Phe Pro Thr Lys Tyr His 365 370 Ser GlyArg Leu Leu Phe Cys Phe Val Pro Gly Asn 375 380 Glu Leu Ile Asp Val SerGly Ile Thr Leu Lys Gln 385 390 395 Ala Thr Thr Ala Pro Cys Ala Val MetAsp Ile Thr 400 405 Gly Val Gln Ser Thr Leu Arg Phe Arg Val Pro Trp 410415 420 Ile Ser Asp Thr Pro Tyr Arg Val Asn Arg Tyr Thr 425 430 Lys SerAla His Gln Lys Gly Glu Tyr Thr Ala Ile 435 440 Gly Lys Leu Ile Val TyrCys Tyr Asn Arg Leu Thr 445 450 455 Ser Pro Ser Asn Val Ala Ser His ValArg Val Asn 460 465 Val Tyr Leu Ser Ala Ile Asn Leu Glu Cys Phe Ala 470475 480 Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val 485 490 Gly AspAsp Ser Gly Gly Phe Ser Thr Thr Val Ser 495 500 Thr Glu Gln Asn Val ProAsp Pro Gln Val Gly Ile 505 510 515 Thr Thr Met Lys Asp Leu Lys Gly LysAla Asn Arg 520 525 Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Val 530535 540 Gly Ala Ile Thr Thr Ile Glu Asp Pro Val Leu Ala 545 550 Lys LysVal Pro Glu Thr Phe Pro Glu Leu Lys Pro 555 560 Gly Glu Ser Arg His ThrSer Asp His Met Ser Ile 565 570 575 Tyr Lys Phe Met Gly Arg Ser His PheLeu Cys Thr 580 585 Phe Thr Phe Asn Ser Asn Asn Lys Glu Tyr Thr Phe 590595 600 Pro Ile Thr Leu Ser Ser Thr Ser Asn Pro Pro His 605 610 Gly LeuPro Ser Thr Leu Arg Trp Phe Phe Asn Leu 615 620 Phe Gln Leu Tyr Arg GlyPro Leu Asp Leu Thr Ile 625 630 635 Ile Ile Thr Gly Ala Thr Asp Val AspGly Met Ala 640 645 Trp Phe Thr Pro Val Gly Leu Ala Val Asp Thr Pro 650655 660 Trp Val Glu Lys Glu Ser Ala Leu Ser Ile Asp Tyr 665 670 Lys ThrAla Leu Gly Ala Val Arg Phe Asn Thr Arg 675 680 Arg Thr Gly Asn Ile GlnIle Arg Leu Pro Trp Tyr 685 690 695 Ser Tyr Leu Tyr Ala Val Ser Gly AlaLeu Asp Gly 700 705 Leu Gly Asp Lys Thr Asp Ser Thr Phe Gly Leu Val 710715 720 Ser Ile Gln Ile Ala Asn Tyr Asn His Ser Asp Glu 725 730 Tyr LeuSer Phe Ser Cys Tyr Leu Ser Val Thr Glu 735 740 Gln Ser Glu Phe Tyr PhePro Arg Ala Pro Leu Asn 745 750 755 Ser Asn Ala Met Leu Ser Thr Glu SerMet Met Ser 760 765 Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp 770775 780 Asp Pro Arg Ser Glu Glu Asp Lys Arg Phe Glu Ser 785 790 His IleGlu Cys Arg Lys Pro Tyr Lys Glu Leu Arg 795 800 Leu Glu Val Gly Lys GlnArg Leu Lys Tyr Ala Gln 805 810 815 Glu Glu Leu Ser Asn Glu Val Leu ProPro Pro Arg 820 825 Lys Met Lys Gly Leu Phe Ser Gln Ala Lys Ile Ser 830835 840 Leu Phe Tyr Thr Glu Glu His Glu Ile Met Lys Phe 845 850 Ser TrpArg Gly Val Thr Ala Asp Thr Arg Ala Leu 855 860 Arg Arg Phe Gly Phe SerLeu Ala Ala Gly Arg Ser 865 870 875 Val Trp Thr Leu Glu Met Asp Ala GlyVal Leu Thr 880 885 Gly Arg Leu Ile Arg Leu Asn Asp Glu Lys Trp Thr 890895 900 Glu Met Lys Asp Asp Lys Ile Val Ser Leu Ile Glu 905 910 Lys PheThr Ser Asn Lys Tyr Trp Ser Lys Val Asn 915 920 Phe Pro His Gly Met LeuAsp Leu Glu Glu Ile Ala 925 930 935 Ala Asn Ser Lys Asp Phe Pro Asn MetSer Glu Thr 940 945 Asp Leu Cys Phe Leu Leu His Trp Leu Asn Pro Lys 950955 960 Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser 965 970 Gly ValGln Glu Ile Lys Glu Gln Gly Val Gly Leu 975 980 Ile Ala Glu Cys Arg ThrPhe Leu Asp Ser Ile Ala 985 990 995 Gly Thr Leu Lys Ser Met Met Phe GlyPhe His His 1000 1005 Ser Val Thr Val Glu Ile Ile Asn Thr Val Leu Cys1010 1015 1020 Phe Val Lys Ser Gly Ile Leu Leu Tyr Val Ile Gln 1025 1030Gln Leu Asn Gln Asp Glu His Ser His Ile Ile Gly 1035 1040 Leu Leu ArgVal Met Asn Tyr Ala Asp Ile Gly Cys 1045 1050 1055 Ser Val Ile Ser CysGly Lys Val Phe Ser Lys Met 1060 1065 Leu Glu Thr Val Phe Asn Trp GlnMet Asp Ser Arg 1070 1075 1080 Met Met Glu Leu Arg Thr Gln Ser Phe SerAsn Trp 1085 1090 Leu Arg Asp Ile Cys Ser Gly Ile Thr Ile Phe Lys 10951100 Asn Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys 1105 1110 1115 LeuLys Asp Phe Tyr Glu Val Asn Tyr Gly Lys Lys 1120 1125 Lys Asp Ile LeuAsn Ile Leu Lys Asp Asn Gln Gln 1130 1135 1140 Lys Ile Glu Lys Ala IleGlu Glu Ala Asp Glu Phe 1145 1150 Cys Ile Leu Gln Ile Gln Asp Val GluLys Phe Glu 1155 1160 Gln Tyr Gln Lys Gly Val Asp Leu Ile Gln Lys Leu1165 1170 1175 Arg Thr Val His Ser Met Ala Gln Val Asp Pro Asn 1180 1185Leu Met Val His Leu Ser Pro Leu Arg Asp Cys Ile 1190 1195 1200 Ala ArgVal His Gln Lys Leu Lys Asn Leu Gly Ser 1205 1210 Ile Asn Gln Ala MetVal Thr Arg Cys Glu Pro Val 1215 1220 Val Cys Tyr Leu Tyr Gly Lys ArgGly Gly Gly Lys 1225 1230 1235 Ser Leu Thr Ser Ile Ala Leu Ala Thr LysIle Cys 1240 1245 Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr 12501255 1260 Thr Lys Pro Val Ala Ser Asp Tyr Trp Asp Gly Tyr 1265 1270 SerGly Gln Leu Val Cys Ile Ile Asp Asp Ile Gly 1275 1280 Gln Asn Thr ThrAsp Glu Asp Trp Ser Asp Phe Cys 1285 1290 1295 Gln Leu Val Ser Gly CysPro Met Arg Leu Asn Met 1300 1305 Ala Ser Leu Glu Glu Lys Gly Arg HisPhe Ser Ser 1310 1315 1320 Pro Phe Ile Ile Ala Thr Ser Asn Trp Ser AsnPro 1325 1330 Ser Pro Lys Thr Val Tyr Val Lys Glu Ala Ile Asp 1335 1340Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala 1345 1350 1355 Ser PhePhe Lys Asn Pro His Asn Asp Met Leu Asn 1360 1365 Val Asn Leu Ala LysThr Asn Asp Ala Ile Lys Asp 1370 1375 1380 Met Ser Cys Val Asp Leu IleMet Asp Gly His Asn 1385 1390 Val Ser Leu Met Asp Leu Leu Ser Ser LeuVal Met 1395 1400 Thr Val Glu Ile Arg Lys Gln Asn Met Thr Glu Phe 14051410 1415 Met Glu Leu Trp Ser Gln Gly Ile Ser Asp Asp Asp 1420 1425 AsnAsp Ser Ala Val Ala Glu Phe Phe Gln Ser Phe 1430 1435 1440 Pro Ser GlyGlu Pro Ser Asn Ser Lys Leu Ser Gly 1445 1450 Phe Phe Gln Ser Val ThrAsn His Lys Trp Val Ala 1455 1460 Val Gly Ala Ala Val Gly Ile Leu GlyVal Leu Val 1465 1470 1475 Gly Gly Trp Phe Val Tyr Lys His Phe Ser ArgLys 1480 1485 Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr His 1490 14951500 Gly Val Thr Lys Pro Lys Gln Val Ile Lys Leu Asp 1505 1510 Ala AspPro Val Glu Ser Gln Ser Thr Leu Glu Ile 1515 1520 Ala Gly Leu Val ArgLys Asn Leu Val Gln Phe Gly 1525 1530 1535 Val Gly Glu Lys Asn Gly CysVal Arg Trp Val Met 1540 1545 Asn Ala Leu Gly Val Lys Asp Asp Trp LeuLeu Val 1550 1555 1560 Pro Ser His Ala Tyr Lys Phe Glu Lys Asp Tyr Glu1565 1570 Met Met Glu Phe Tyr Phe Asn Arg Gly Gly Thr Tyr 1575 1580 TyrSer Ile Ser Ala Gly Asn Val Val Ile Gln Ser 1585 1590 1595 Leu Asp ValGly Phe Gln Asp Val Val Leu Met Lys 1600 1605 Val Pro Thr Ile Pro LysPhe Arg Asp Ile Thr Gln 1610 1615 1620 His Phe Ile Lys Lys Gly Asp ValPro Arg Ala Leu 1625 1630 Asn Arg Leu Ala Thr Leu Val Thr Thr Val AsnGly 1635 1640 Thr Pro Met Leu Ile Ser Glu Gly Pro Leu Lys Met 1645 16501655 Glu Glu Lys Ala Thr Tyr Val His Lys Lys Asn Asp 1660 1665 Gly ThrThr Val Asp Leu Thr Val Asp Gln Ala Trp 1670 1675 1680 Arg Gly Lys GlyGlu Gly Leu Pro Gly Met Cys Gly 1685 1690 Gly Ala Leu Val Ser Ser AsnGln Ser Ile Gln Asn 1695 1700 Ala Ile Leu Gly Ile His Val Ala Gly GlyAsn Ser 1705 1710 1715 Ile Leu Val Ala Lys Leu Val Thr Gln Glu Met Phe1720 1725 Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile 1730 1735 1740Met Lys Val Glu Phe Thr Gln Cys Ser Met Asn Val 1745 1750 Val Ser LysThr Leu Phe Arg Lys Ser Pro Ile Tyr 1755 1760 His His Ile Asp Lys ThrMet Ile Asn Phe Pro Ala 1765 1770 1775 Ala Met Pro Phe Ser Lys Ala GluIle Asp Pro Met 1780 1785 Ala Val Met Leu Ser Lys Tyr Ser Leu Pro IleVal 1790 1795 1800 Glu Glu Pro Glu Asp Tyr Lys Glu Ala Ser Ile Phe 18051810 Tyr Gln Asn Lys Ile Val Gly Lys Thr Gln Leu Val 1815 1820 Asp AspPhe Leu Asp Leu Asp Met Ala Ile Thr Gly 1825 1830 1835 Ala Pro Gly IleAsp Ala Ile Asn Met Asp Ser Ser 1840 1845 Pro Gly Phe Pro Tyr Val GlnGlu Lys Leu Thr Lys 1850 1855 1860 Arg Asp Leu Ile Trp Leu Asp Glu AsnGly Leu Leu 1865 1870 Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu1875 1880 Phe Asn Thr Val Met Met Glu Asn Cys Ser Asp Leu 1885 1890 1895Asp Val Val Phe Thr Thr Cys Pro Lys Asp Glu Leu 1900 1905 Arg Pro LeuGlu Lys Val Leu Glu Ser Lys Thr Arg 1910 1915 1920 Ala Ile Asp Ala CysPro Leu Asp Tyr Ser Ile Leu 1925 1930 Cys Arg Met Tyr Trp Gly Pro AlaIle Ser Tyr Phe 1935 1940 His Leu Asn Pro Gly Phe His Thr Gly Val AlaIle 1945 1950 1955 Gly Ile Asp Pro Asp Arg Gln Trp Asp Glu Leu Phe 19601965 Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp 1970 1975 1980 LeuAsp Phe Ser Ala Phe Asp Ala Ser Leu Ser Pro 1985 1990 Phe Met Ile ArgGlu Ala Gly Arg Ile Met Ser Glu 1995 2000 Leu Ser Gly Thr Pro Ser HisPhe Gly Thr Ala Leu 2005 2010 2015 Ile Asn Thr Ile Ile Tyr Ser Lys HisLeu Leu Tyr 2020 2025 Asn Cys Cys Tyr His Val Cys Gly Ser Met Pro Ser2030 2035 2040 Gly Ser Pro Cys Thr Ala Leu Leu Asn Ser Ile Ile 2045 2050Asn Asn Val Asn Leu Tyr Tyr Val Phe Ser Lys Ile 2055 2060 Phe Gly LysSer Pro Val Phe Phe Cys Gln Ala Leu 2065 2070 2075 Lys Ile Leu Cys TyrGly Asp Asp Val Leu Ile Val 2080 2085 Phe Ser Arg Asp Val Gln Ile AspAsn Leu Asp Leu 2090 2095 2100 Ile Gly Gln Lys Ile Val Asp Glu Phe LysLys Leu 2105 2110 Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro 21152120 Gln Leu Lys Pro Val Ser Glu Leu Thr Phe Leu Lys 2125 2130 2135 ArgSer Phe Asn Leu Val Glu Asp Arg Ile Arg Pro 2140 2145 Ala Ile Ser GluLys Thr Ile Trp Ser Leu Ile Ala 2150 2155 2160 Trp Gln Arg Ser Asn AlaGlu Phe Glu Gln Asn Leu 2165 2170 Glu Asn Ala Gln Trp Phe Ala Phe MetHis Gly Tyr 2175 2180 Glu Phe Tyr Gln Lys Phe Tyr Tyr Phe Val Gln Ser2185 2190 2195 Cys Leu Glu Lys Glu Met Ile Glu Tyr Arg Leu Lys 2200 2205Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln 2210 2215 2220 Cys PheIle Cys Asp Leu Ser 2225 7400 base pairs nucleic acid single linear notprovided 3 CTTGATACCT CACCGCCGTT TGCCTAGGCT ATAGGCTTCT 40 TCCCTACACCCTTGTTTGTT TTTTTTTTTT TTTTTTGTGT 80 GTTTGTAAAT ATTAATTCCT GCAGGTTCAGGGTTCTTAAT 120 TTGTTCTGCT ATACAGACAC TCTTTTCACG CTTTCTGTCA 160TCTTATTTCC TGGGCTCTCC CCTTGCCCAA GGCTCTGGCC 200 GTTGCGCCCG GCGGGGTCAACTCCATGGTT AGCATGGAGC 240 TGTAGGAGTC TAAATTGGGG ACGCAGATGC TAGGAACGTC280 GCCCTGCAGT GTTAACCTGG CTTTCATGAA GCTCTTTGAT 320 CTTCTACAAGAGGTAGGCTA CGGGTGAAAC CTCTTAGATT 360 AATACTCCTA TGGAGAGATA TCTTGAATAGGGTAACAGCG 400 GTGGATATTG GTGAGTTCCT TTGGGACAAA AACCATTCAA 440CACCGGAGGA CTGACTCTCA TTCAGTAGTT GCATTGAGTG 480 AATTGTCTGT CAGGGCTGTCTTTGGGTTTA ATTCCTGGCC 520 TCTCTGTGCT TAGGGCAAAC CATTTCCTGG CCTTAAATGG560 AGTTCTGTGA GAGGGAACTC CTCCTTTATA TGCTGGACAT 600 ATTTTGGGGCCTTAGGGTTA TGGTTTGCCT CTGAGGTACT 640 CAGGGGCATT TAGGTTTTTC CTCATTTATATGTTTATGAT 680 GATGAATATG TCTAAACAAG GTATTTTCCA GACTGTTGGG 720AGTGGCCTTG ACCACATACT GTCTTTAGCA GATGTGGAGG 760 AAGAGCAAAT GATACAGTCAGTGGACAGGA CAGCTGTCAC 800 TGGTGCTTCT TATTTTACTT CTGTAGACCA ATCTTCAGTT840 CATACGGCAG AAGTTGGTGC ACATCAGACA GAGCCTCTTA 880 AGACATCAGTAGATAAACCA GGTTCAAAGA AAACCCAAGG 920 AGAGAAATTT TTCCTAATAC ATTCTGCAGATTGGTTAACA 960 ACACATGCTT TGTTTCATGA AGTCGCCAAA TTGGATGTTG 1000TTAGTTTGTT GTACAATGAA CAATTTGCTG TACAGGGTTT 1040 GTTGAGATAC CATACTTATGCTAGATTTGG AATTGAAATT 1080 CAAGTCCAGA TTAATCCCAC TCCCTTTCAG CAGGGAGGTC1120 TTATTTGTGC AATGGTTCCA GGAGACCAAG GTTATGGTTC 1160 CATAGCCTCATTGACAGTTT ATCCACATGG TCTCTTGAAT 1200 TGCAACATTA ACAATGTTGT TAGAATCAAAGTTCCATTCA 1240 TTTATACTAG AGGTGCTTAT CATTTCAAAG ATCCACAGTA 1280TCCAGTCTGG GAGTTAACTA TTCGTGTTTG GTCAGAATTA 1320 AATATAGGAA CTGGTACTTCTGCTTATACA TCATTGAATG 1360 TCTTGGCTAG ATTCACTGAT TTAGAGCTTC ATGGATTGAC1400 ACCATTATCT ACACAAATGA TGAGGAATGA ATTTAGAGTG 1440 AGTACAACTGAAAATGTGGT TAATTTGTCA AATTACGAGG 1480 ATGCTAGAGC AAAGATGTCT TTTGCACTTGATCAGGAAGA 1520 TTGGAAAACA GATCCCTCGC AAGGAGGAGG AATCAAAATC 1560ACTCATTTTA CAACATGGAC TTCAATTCCC ACGCTTGCTG 1600 CACAGTTTGC ATTTAATGCTTCTGCATCTG TGGGGCAGCA 1640 AATTAAGGTG ATCCCTGTTG ATCCTTATTT TTATCAGATG1680 ACCAATTCAA ATCCAGACCA AAAGTGTATT ACTGCTTTAG 1720 CTTCTGTCTGTCAGATGTTC TGCTTTTGGA GGGGAGATCT 1760 TGTTTTTGAT TTTCAGGTTT TCCCCACAAAATATCACTCT 1800 GGGAGGTTGT TATTTTGTTT TGTGCCAGGG AATGAGTTGA 1840TAGATGTTTC AGGTATAACC CTGAAGCAGG CAACTACTGC 1880 ACCCTGTGCT GTTATGGATATAACAGGAGT TCAGTCAACA 1920 TTGAGATTTA GAGTGCCTTG GATCTCTGAT ACACCTTACA1960 GAGTGAATAG ATACACAAAA TCAGCTCACC AGAAAGGAGA 2000 GTATACAGCTATTGGGAAGT TGATTGTTTA TTGTTATAAT 2040 AGGCTTACCT CACCCTCAAA TGTTGCTTCCCATGTTAGGG 2080 TTAATGTTTA TCTTTCTGCA ATAAATTTGG AATGTTTTGC 2120ACCCCTATAT CATGCAATGG ATGTGACATC ACAGACAGGT 2160 GATGATTCAG GTGGGTTTTCAACTACAGTT TCTACAGAAC 2200 AGAATGCTCC TGATCCTCAA GTTGGAATTA CCACTATTAA2240 GGATTTAAAA GGGAAGGCAA ATAGAGGAAA GATGGATGTT 2280 TCTGGCATTCAAGCACCAGT GGGTGCTATT ACAACCATTG 2320 AGGATCCAGT GTTAGCTAAA AAAGTTCCTGAGACTTTTCC 2360 AGAATTGAGA CCAGGTGAAT CTAGACATAC TTCAGATCAT 2400ATGTCTATTT ACAAATTTAT GGGGAGGTCA CACTTTCTTT 2440 GTACATTTAC TTTCAATGCAAACAATAGGG AGTATACTTT 2480 TCCAATAACA CTGTCCTCTA CATCGAATCC ACCTCATGGT2520 TTACCATCAA CACTGAGGTG GTTTTTCAAC CTTTTTCAAT 2560 TGTATAGAGGGCCATTGGAC TTGACTATTA TAATTACAGG 2600 TGCTACTGAT GTGGATGGCA TGGCTTGGTTTACTCCTGTG 2640 GGCCTAGCTG TGGATACTCC CTGGGTTGAA AAGCAATCAG 2680CGTTGACTAT TGATTATAAA ACTGCTCTTG GGGCTATTAG 2720 GTTTAACACT AGGAGAACAGGAAATATTCA GATTAGACTT 2760 CCTTGGTATT CATACCTTTA TGCTGTTTCT GGCGCTTTGG2800 ATGGACTTGG GGACACTACT GATTCGACTT TCGGGTTGGT 2840 CTCTATTCAGATTGCCAATT ATAATCATTC AGATGAATAT 2880 CTGTCATTCA GTTGTTATCT TTCAGTTACTGAACAATCAG 2920 AATTTTATTT TCCAAGGGCT CCTCTCAATT CTAATGCTAT 2960GATGGTTTCT GAGTCCATGC TAGATCGCAT TGCAAGTGGA 3000 GATTTAGAAT CATCAGTTGATGACCCAAGA TCAGCAGAGG 3040 ACAAAAGGTT TGAAAGTCAT ATTGAGCAGG GCAAGCCATA3080 CAAAGAATTA AGAATGGAAG TTGGGAAGCA GAGATTGAAA 3120 TATGCCATGGAGGAGTTATC AAATGAAATT TTACCACCTC 3160 CTCGGAAAGT GAAAGGACTG TTTTCTCAAGCTAAAATTTC 3200 TTTATTTTAT ACAGAAGACC ATGAAATTGT GAAGCTTTCA 3240TGGAAAGGTC TCACAGCTGA TACAAGAGCT CTCAGGAGAT 3280 ATGGTTTTTC TCTTGCTGCTGGAAGAAGTG TGTGGACTCT 3320 TGAGATGGAA GCTGGAGTTC TGACTGGAAG GATGATCAGA3360 TTGAATGATG AAAAGTGGAC TGAGATTAAG GATGATAAGA 3400 TAGTGGCTTTGGTAGAGAAA TTTACATCTA ATAAGAATTG 3440 GTCTAAAGTC AATTTTCCAC ATGGGATGCTAGATTTGGAA 3480 GAGATAGCAT CAAATTCAAA GGATTTTCCT AATATGTCTG 3520AGACTGACTT GTGTTTTCTT TTACATTGGT TGAATCCTAA 3560 GAAGATAAAT CTAGCTGATAGAATGCTTGG ATTGTCTGGT 3600 GTTCAGGAAA TTAAGGAACA GGGTGTTGGC TTAATAGCTG3640 AATGTAGAAC ATTTTTAGAT TCTATAGCTG GCACTTTGAA 3680 ATCAATGATGTTTGGGTTTC ATCAGTCTGT TACTGTGGAA 3720 ATAATTAATA CTGTCTTGTG TTTTGTTAAGAGTGGGATCC 3760 TTCTTTATGT TATTCAGCAA TTGAATCAAA ATGAACACTC 3800TCATATTATA GGGCTTTTAC AGGTGATGAA TTATGCAGAC 3840 ATTGGTTGCT CTGTGATTTCTTGTGGAAAG ATATTCTCAA 3880 AAATGTTAGA AACAGTCTTT AATTGGCAGA TGGATTCTAG3920 AATGATGGCT CTTAGAACAC AGAGTTTCTC TAATTGGTTG 3960 AGAGACATATGTTCGGGGAT AACCATTTTC AAAAATTTTA 4000 AGGATGCTAT TTTCTGGCTG TACACTAAATTAAAGGATTA 4040 TTATGATTCT AACTATGGGA AAAAGAAGGA TGTTCTGAAT 4080GTTTTAAAAG AAAATCAGCA TAGGATTGAG AAAGCCATTG 4120 AAGAGGCTGA TCAGTTCTGTGTTTTGCAGA TTCAGGACGT 4160 TGAGAAGTCA GAGCAATATC AGAAGGGAGT TGAACTCATT4200 CAGAAATTGA GAACAGTTCA TTCCCTGGCC CAGGTCGACT 4240 CTAGTTTGATGTCTCATTTG TCACCACTGA GAGATTGTAT 4280 TGCTAGAGTC CATCAAAAAC TTAAGAATTTAGGCTCAATT 4320 AATCAGGCTA TGGTGACTAG GTGTGAACCT GTGGTCTGTT 4360ATTTATATGG TAAGAGAGGT GGAGGAAAGA GTTTAACTTC 4400 TATTGCATTG GCAACAAAAATTTGCAAACA TTATGGTGTT 4440 GAACCAGAAA AGAATATATA TACAAAACCT GTTGCTTCAG4480 ACTACTGGGA TGGATATAGT GGTCAATTGG TTTGTATCAT 4520 TGATGACATTGGTCAAAATA CTACAGATGA AGATTGGTCA 4560 GATTTTTGTC AATTGGTGTC TGGTTGTCCTATGAGGTTAA 4600 ATATGGCTTC TTTGGAAGAG AAAGGGAGAC ACTTTTCTTC 4640CCCGTTTATA ATTGCCACAT CAAATTGGTC AAATCCAAGT 4680 CCTAAGACTG TTTATGTGAAGGAAGCTATA GATCGCCGCC 4720 TTCATTATAA GATTGAAGTC AAACCAGCAT CTTTTTACAA4760 AAATGCACAC AATGATATGC TCAATGTGAA TCTTGCAAGA 4800 AATAATGATGCCATTAAAGA CATGTCCTGT GTAGATTTAC 4840 TGATGGATGG CCATACTGTG TCTTTATCTGAGCTTTTAAA 4880 TTCTCTTGTT ATGACAGTTG AAATTAGAAA ACAAAATATG 4920TCAGAATTTA TGAAATTGTG GTCACAGGGT GTGTCAGATG 4960 ATGATAATGA CAGTGCAGTTGCTGAGTTCT TCCAGTCTTT 5000 TCCATCAGGA GAACCCTCAA ATTCTAAGTT ATCTAGTTTC5040 TTCAAGGCGG TCACTAATCA TAAGTGGGTT GCTATTGGAG 5080 CTGCTGTTGGAGTTCTGGGT GTCTTAGTGG GAGGTTGGTT 5120 TGTGTACAAG CATTTTACCA AAGAAGAACCAATACCAACT 5160 GAAGGAGTGT ATCATGGAGT AACCAAACCT AAACAGGTTA 5200TCAAATTGGA TGCTGATCCT GTTGACTCCC AATCTACTCT 5240 TGAGATAGCT GGACTAGTTAGGAAGAATTT GGTTCAATTT 5280 GGAGTTGGGG AGAAGAATGG ATGTGTTAGG TGGGTCATGA5320 ATGCTTTAGG TATTAAAGAT GATTGGCTGC TGGTCCCCTC 5360 ACATGCATACAAATTTGAGA AAGATTATCA AATGATGGAG 5400 TTTTATTTTA ATAGAGGAGG AACTTATTATTCAATTTCTG 5440 CTGGTAATGT TGTAATCCAG TCTTTGGATG TTGGTTTTCA 5480GGATGTTGTT TTGATGAAGG TTCCTACAAT TCCAAAGTTT 5520 AGAGATATAA CTGAGCATTTTATTAAGAAG AATGATGTTC 5560 CAAGAGCTTT GAATAGATTG GCTACACTTG TTACAACAGT5600 TAATGGGACA CCAATGCTGA TTTCCGAAGG TCCACTTAAG 5640 ATGGAAGAAAAGGCCACTTA TGTCCATAAG AGAAATGACG 5680 GAACTACTGT TGATTTGACT GTTGATCAAGCTTGGAGGGG 5720 AAAAGGTGAG GGCCTCCCAG GTATGTGTGG TGGAGCTCTG 5760ATTTCCTCAA ATCAGTCAAT ACAAAATGCC ATTCTTGGGA 5800 TTCATGTTGC AGGTGGCAATTCTATTTTGG TTGCCAAACT 5840 TGTGACTCAG GAAATGTTCC AGAACATTGA ACAAAAAGCA5880 ATAGAAAGTC AGAGGATAAT GAAAGTGGAA TTCACTCAGT 5920 GTTCAATGAATGTGGTCTCC AAAACGCTTT TTAAAAAGAG 5960 TCCAATTCAT CATCACATTG ATAGGAACATGATTAATTTT 6000 CCTGCTGTAA TGCCTTTTTC TAAAGCTGAG ATTGATCCTA 6040TGGCTGTTAT GTTGTCTAAG TATTCTCTTC CTATTGTTGA 6080 AGAGCCAGAT GATTATAAGATGGCTTCCAT TTATTTCCAA 6120 AATAAAGTAA TGGGGAAAAC TTTTCTTGTT GATGACTTTT6160 TGGATATAGA TATGGCAATC ACAGGTGCTC CAGGAATAGA 6200 TGCTATTAATATGGATTCTT CACCAGGATT TCCTTATGTT 6240 CAGGAGAAGT TGACAAAGAA AGACTTGATCTGGTTGGATG 6280 AGAATGGGCT GCTGTTAGGA GTTCATCCAA GGCTTGCTCA 6320AAGAATCTTG TACAACACAG TTATGATGGA GAATTGTTCT 6360 GATCTTGATG TGGTCTTTACAACATGTCCC AAGGATGAAC 6400 TTAGGCCTCT GGACAAAGTA TTGGAATCAA AGACTAGAGC6440 AATTGATGCT TGTCCATTGG ATTATACAAT TCTTTGTAGG 6480 ATTTATTGGGGTCCTGCTAT TAGTTACTTT CAATTGAATC 6520 CTGGATTTCA CACAGGAGTT GCTATTGGAATTGATCCGGA 6560 TAGACATTGG GACGAGTTGT TTAAAACAAT GGTTAGATTT 6600GGTGATGTAG GTTTAGACCT TGATTTTTCA TCATTTGATG 6640 CTAGTCTTAG TCCTTTTATGATAAGAGAGG CAGGGAGAAT 6680 TTTGAGTGAA ATGTCAGGGA CACCCTCACA CTTTGGAGAG6720 GCCTTGATTA ATACAATCAT TTATTCCAAG CATTTGTTGT 6760 ACAATTGTTGTTATCATGTT TATGGTTCCA TGCCATCAGG 6800 GTCCCCTTGT ACAGCACTTT TAAATTCAATTGTAAACAAT 6840 GTTAATTTGT ACTATGTGTT TTCAAAAATT TTTAGGAAGT 6880CTCCTGTTTT CTTTGGAGAT GCTCTGAAGA TTCTTTGTTA 6920 TGGAGATGAT GTCCTCATTGTTTTTTCCAG AAATGTCCAG 6960 ATTGATAATT TGGAATCTAT TGGACAGAAA ATTGTAGATG7000 AGTTTGGAAA ATTAGGCATG ACTGCAACAT CAGCAGACAA 7040 GTCTGTTCCTAAGTTGAAAC CTATTTCTGA GCTCACTTTT 7080 CTTAAAAGAT CATTCAATCT TGTTGAAGATCGGATTAGAC 7120 CTGCAATTTC AGAGAAAACA ATTTGGTCTC TCGTTGCTTG 7160GCAGAGAAGC AATGCTGAAT TTGAACAGAA TTTGGAAAAT 7200 GCTCAATGGT TTGCTTTTATGCATGGTTTT GAATTTTATC 7240 AGAAATTTTA CCATTTTGTT CAGTCCTGCC TGGAGAAAGA7280 GATGGTAGAA TACAGATTGA AATCATATGA TTGGTGGAGA 7320 ATGAAGTTTTATGATCAGTG CTTTGTTTGT GACCTCACAT 7360 GATTTGTTTA AACAAACCTT CTTAAAATTTCTGAGATTTG 7400

What is claimed is:
 1. A DNA construct comprising a whole humanattenuated hepatitis A virus (HAV) genome sequence wherein at least onefragment of the 2C gene of the human attenuated genome sequence isreplaced by the corresponding fragment(s) of the 2C gene of a simianhepatitis A virus AGM-27 genome sequence, wherein the AGM-27 2C genefragment does not comprise the entire 2C gene and encodes an amino acidsequence which differs in at least one amino acid residue from the aminoacid sequence encoded by the corresponding fragment of the 2C gene ofthe human attenuated hepatitis A virus.
 2. The DNA construct of claim 1,wherein the amino acid sequence encoded by the AGM-27 2C gene fragmentdiffers in at least five amino residues from the amino acid sequenceencoded by the corresponding fragment of the 2C gene of the humanattenuated hepatitis A virus.
 3. The DNA construct of claim 1, whereinone fragment of the 2C gene of the human attenuated hepatitis A virusgenome sequence is replaced.
 4. The DNA construct of claim 3, whereinthe AGM-27 2C gene fragment encodes amino acid residues of the 2Cprotein selected from the group consisting of amino acid residues48-328, 63-328, 84-328, 98-328, 1-49, 1-64, 1-86, 1-99, 1-283, 1-294,1-304, 1-120 and 303-328, 1-70, 74-120, 74-328, 120-328, 1-121 and 1-328of the 2C protein.
 5. The DNA construct of claim 4, wherein the AGM-272C gene fragment encodes amino acids 120-328 of the 2C protein.
 6. TheDNA construct of claim 4, wherein the AGM-27 2C gene fragment encodesamino acids 1-121 of the 2C protein.
 7. The DNA construct of claim 4,wherein the AGM-27 2C gene fragment encodes amino acids 1-328 of the 2Cprotein.
 8. The DNA construct of claim 1, wherein two fragments of the2C gene of the human attenuated HAV genome sequence are replaced.
 9. TheDNA construct of claim 8, wherein the two fragments encode amino acidresidues of the 2C protein selected from the group of fragmentsconsisting of amino acid residues 1-49 and 121-328, 1-64 and 121-328,1-86 and 121-328, 1-99 and 121-328, 1-120 and 283-328, and 1-120 and294-328 of the 2C protein.
 10. An RNA transcript of the DNA construct ofclaim
 1. 11. A cell transfected with the DNA construct of claim
 1. 12. Acell transfected with the RNA transcript of claim
 10. 13. A hepatitis Avirus having a genome comprising a human attenuated hepatitis A virusgenome in which at least one fragment of the 2C gene of the attenuatedhepatitis A virus genome has been replaced by a corresponding fragmentof a 2C gene from a simian AGM-27 hepatitis A virus genome sequence,wherein the AGM-27 2C gene fragment does not comprise the entire 2C geneand encodes an amino acid sequence which differs in at least one aminoacid residue from the amino acid sequence encoded by the correspondingfragment of the 2C gene of the human attenuated HAV.
 14. The hepatitis Avirus of claim 13, wherein the amino acid sequence encoded by the AGM-272C gene fragment differs in at least five amino residues from the aminoacid sequence encoded by the corresponding fragment of the 2C gene ofthe human attenuated HAV.
 15. The hepatitis A virus of claim 13, whereinone fragment of the 2C gene of the human attenuated HAV is replaced. 16.The hepatitis A virus of claim 15, wherein the AGM-27 2C gene fragmentencodes amino acid residues of the 2C protein selected from the groupconsisting of amino acid residues 48-328, 63-328, 84-328, 98-328, 1-49,1-64, 1-86, 1-99, 1-283, 1-294, 1-304, 1-120 and 303-328, 1-70, 74-120,74-328, 120-328, 1-121 and 1-328 of the 2C protein.
 17. The hepatitis Avirus of claim 16, wherein the AGM-27 2C gene fragment encodes aminoacids 120-328 of the 2C protein.
 18. The hepatitis A virus of claim 16,wherein the AGM-27 2C gene fragment encodes amino acids 1-121 of the 2Cprotein.
 19. The hepatitis A virus of claim 16, wherein the AGM-27 2Cgene fragment encodes amino acids 1-328 of the 2C protein.
 20. Thehepatitis A virus of claim 13, wherein two fragments of the 2C gene ofthe human attenuated HAV are replaced.
 21. The hepatitis A virus ofclaim 20, wherein the two fragments encode amino acid residues of the 2Cprotein selected from the group consisting of amino acid residues 1-49and 121-328, 1-64 and 121-328, 1-86 and 121-328, 1-99 and 121-328, 1-120and 283-328, and 1-120 and 294-328 of the 2C protein.
 22. A vaccine forpreventing hepatitis A in a mammal, said vaccine comprising the DNAconstruct of claim 1 and a pharmaceutically acceptable carrier.
 23. Avaccine for preventing hepatitis A in a mammal, said vaccine comprisingthe RNA transcript of claim 10 and a pharmaceutically acceptablecarrier.
 24. A vaccine for preventing hepatitis A in a mammal, saidvaccine comprising the hepatitis A virus of claim 13 and apharmaceutically acceptable carrier.
 25. A method for preventinghepatitis A virus in a mammal, said method comprising administering tosaid mammal the DNA construct of claim 1 in an amount effective tostimulate the production of protective antibodies in said mammal.
 26. Amethod of preventing hepatitis A virus in a mammal, said methodcomprising administering to said mammal the RNA transcript of claim 10in an amount effective to stimulate the production of protectiveantibodies in said mammal.
 27. A method of preventing hepatitis A virusin a mammal, said method comprising administering to said mammal thehepatitis A virus of claim 13 in an amount effective to stimulate theproduction of protective antibodies in said mammal.
 28. A pharmaceuticalcomposition comprising the DNA construct of claim
 1. 29. Apharmaceutical composition comprising the RNA transcript of claim 10.30. A kit for the prevention of hepatitis A in a mammal, said kitcomprising the DNA construct of claim
 1. 31. A kit for the prevention ofhepatitis A in a mammal, said kit comprising the RNA transcript of claim10.
 32. A kit for the prevention of hepatitis A in a mammal, said kitcomprising the hepatitis A virus of claim
 13. 33. A host cell containingthe hepatitis A virus of claim 13.